J Neurooncol (2012) 107:479–485 DOI 10.1007/s11060-011-0775-2
LABORATORY INVESTIGATION - HUMAN/ANIMAL TISSUE
F10 gene hypomethylation, a putative biomarker for glioma prognosis Xiaoping Liu • Hailin Tang • Zeyou Wang • Chen Huang • Zuping Zhang • Xiaoling She Minghua Wu • Guiyuan Li
•
Received: 14 March 2011 / Accepted: 29 November 2011 / Published online: 13 December 2011 Ó Springer Science+Business Media, LLC. 2011
Abstract Tumors are usually characterized by an imbalance in cytosine methylation, including hypomethylation of CpG islands. In this study, bisulfite sequencing PCR was used to assess the promoter methylation status of coagulation factor X (F10) gene in tumors of 96 glioma patients and in glioma cells U251, SF767, and SF126, and the effect of promoter hypomethylation on protein expression was evaluated immunohistochemically. The study showed that the demethylation ratio of F10 in SF126, SF767, and U251 cells was 38.6, 26.4, and 24.3% respectively. Hypomethylation of F10 was detected in 82.3% of glioma specimens and in no normal brain tissues, with significant correlation with its protein expression. However
there was no remarkable relationship between F10 hypomethylation and sex, age, and advanced tumor grade. The correlation between F10 hypomethylation, protein expression, and overall survival (OS) was statistically significant. Hypomethylation of F10 promoter in gliomas accounted for F10 encoding protein FX overexpression and aggressive biological behavior in a subset of patients. Furthermore, in the F10 hypomethylation group, OS was shorter for patients with F10 overexpression than for those without. Detection of these epigenetic changes in tumors may provide important information regarding prognosis.
X. Liu H. Tang Z. Wang Z. Zhang X. She M. Wu (&) G. Li (&) Cancer Research Institute, Central South University, 110# Xiangya Road, Changsha, Hunan Province, China e-mail:
[email protected]
H. Tang Cancer Research Institute, University of South China, 28# Changsheng West Road, Hengyang 421001, China
G. Li e-mail:
[email protected] X. Liu H. Tang Z. Wang Z. Zhang X. She M. Wu G. Li Disease Genome Research Center, Central South University, Changsha, China X. Liu H. Tang Z. Wang Z. Zhang X. She M. Wu G. Li Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Changsha, China
Keywords
Glioma F10 Hypomethylation Prognosis
C. Huang Medical Research Center, Peking University Third Hospital, Beijing, China Z. Zhang Parasitology Department, Xiangya Medical School, Central South University, 110# Xiangya Road, Changsha, Hunan Province, China X. She Department of Pathology, The Second Xiangya Hospital, Central South University, Changsha, China
X. Liu H. Tang Z. Wang Z. Zhang X. She M. Wu G. Li Key Laboratory of Carcinogenesis, Ministry of Health, Changsha, China
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Introduction
Materials and methods
Gliomas, the most common primary tumors arising in the human central nervous system, are classified into grades I, II, III, or IV by the WHO classification system [1]. Despite recent advances in treatment by a combination of surgery and chemotherapy and/or radiotherapy, the prognosis of malignant glioma is still extremely poor. In the United States 75% die within 5 years of diagnosis [2]. In a series of 320 patients presenting with histologically verified GBM, overall survival (OS) of all patients was 12.3 months. For 91 patients who were reoperated, median OS was 18.9 months, whereas for patients without reoperation median OS was 9.7 months [3]. There is, therefore, an urgent need for development of a prognosis biomarker for this disease. Aberrant epigenetic mechanisms, for example promoter hyper/hypomethylation, histone modifications, or noncoding RNA expression, are important in tumor formation [4]. Changes in DNA methylation patterns are important indicators of tumor development and progression [5]. Reduction in the level of methylation contributes to neoplastic progression in numerous types of human cancer, including glioblastomas [6, 7]. The F10 gene encodes vitamin K-dependent coagulation factor X of the blood coagulation cascade. This factor undergoes multiple processing steps before its preprotein is converted to a mature two-chain form by excision of the tripeptide RKR. The two chains of the factor are held together by one or more disulfide bonds. The light chain carries two EGF-like domains whereas the heavy chain carries the catalytic domain which is structurally homologous with those of the other hemostatic serine proteases. The mature factor is activated by cleavage of the activation peptide by factor IXa (in the intrinsic pathway) or by factor VIIa (in the extrinsic pathway). During blood clotting, prothrombin is converted by the activated factor to thrombin in the presence of factor Va, Ca2?, and phospholipid. Mutations of this gene result in factor X deficiency, a hemorrhagic condition of variable severity [8]. Some researchers have investigated FXa function in tumor cell migration [9, 10]; usually, however, little attention is devoted to F10 gene function in tumors. In this study, the promoter methylation status of F10 in glioma was investigated to determine any correlation between tumors and DNA methylation. Correlation of F10 methylation status with clinical data was investigated to assess its predictive and prognostic value for glioma patients. Expression of F10 in these tumors was also assessed to determine whether promoter hypomethylation correlated with protein overexpression in gliomas. Regular follow-up of these patients and correlation of molecular findings with disease outcome emphasized the prognostic relevance of F10 hypomethylation in glioma patients.
Tissue specimens
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Frozen tissue samples from 96 gliomas and 10 normal brains were obtained from Xiangya Hospital of Central South University, Hunan, China, between January 2009 and June 2011. The study was conducted with the approval of the Ethics Committee of the Faculty of Medicine, Central South University, and after obtaining informed consent from all patients. Normal samples were obtained from autopsies. Tumor samples were diagnosed, by use of the World Health Organization (WHO) system, by two pathologists who were unaware of patient data. Clinical data, including gender, age, initial presentation, postoperative irradiation, chemotherapy, follow-up, and outcome, were obtained from medical records. Forty-two female and 54 male patients ranging in age from 5 to 64 years were included; the mean and median ages were 41.8 and 44 years, respectively. Cell lines and treatments The following human glioma cell lines obtained from the Cell Center of Peking Union Medical College (Beijing, China) were used: U251, SF767, and SF126. U251 cells were maintained in Dulbecco’s modified Eagle medium (Gibco, Germany) and SF767 and SF126 cells were maintained in minimum essential medium (Gibco), with 10% FCS, 100 units/ml penicillin, and 100 lg/ml streptomycin, at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Isolation of genomic DNA from cell lines and tissues Genomic DNA was isolated from the three cell lines, glioma tissues, and normal brain tissues by use of the Universal Genomic DNA Extraction Kit Ver.3.0 (Takara, Dalian, Liaoning, China) in accordance with the manufacturer’s instructions. The quality and integrity of DNA from tissues and cells were checked by electrophoresis on 1% agarose gel, quantified spectrophotometrically, and stored at -20°C until further use. Bisulfite DNA Genomic DNA (0.5 lg) extracted from the cells and from tumor and normal tissue specimens was subject to bisulfite treatment with an Epitect Bisulfite Kit (Qiagen, Hilden, Germany). Briefly, DNA was denatured by treatment with NaOH and modified by sodium bisulfite. DNA samples were then purified on the column provided with the kit,
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again treated with NaOH, precipitated with ethanol, and resuspended in water. Bisulfite sequencing PCR BSP was conducted as described elsewhere [11], commencing with amplification of the bisulfite-treated F10 promoter containing 28 CpG sites, by using two sequential PCR reactions from bisulfite-treated DNA with the primers 5 0 -TTTATTTTTATGTATTAGGGGGTGAG-3 0 (sense) and 50 -TACAAACAATCCTAAATCATCAACC-30 (antisense) (nucleotide location -449 to -4465 and -4135 to -4110, product size 381 bp). For PCR, 2.5 U Taq mix (Takara) and 0.5 ll lM forward and reverse primers were used in a 50 ll total reaction volume. Here 100 ng bisulfite-treated DNA was used as the template for PCR. The PCR cycles were: 95°C for 5 min, followed by 38 cycles of 95°C for 0.5 min, 57°C for 2 min, and 72°C for 2.5 min, with a final extension at 72°C for 10 min. The PCR products were purified by extraction from 1% agarose gel and ligated into the pGEM-T vector (Promega, Madison, WI, USA) in a 3:1 vector-to-PCR product ratio. The ligation products were used to transform competent Escherichia coli cells (strain JM109) by use of standard procedures, and blue/white screening was used to select a minimum of five bacterial transformants (clones). The F10 promoter of positive clones was sequenced by Genscript (Nanjing, China). The methylation decrease for each sample was calculated as the percentage of unmethylated CpG dinucleotides from the total number of CpG dinucleotides been analyzed. Immunohistochemistry Immunohistochemical analysis was carried out with paraffin-embedded glioma and normal brain tissue sections. Briefly, after antigen retrieval by microwave heating in 0.01 M citrate buffer (pH 6.0) for 15 min, tissue sections were rinsed with 0.01 M phosphate-buffered saline, pH 7.4 (PBS). Sections were incubated with primary antibody (1:150 dilution, sc-20673; Santa Cruz Biotechnology, Heidelberg, Germany) at 4°C overnight in a humified chamber. After extensive washing with PBS, sections were incubated with biotin-linked rabbit anti-goat IgG antibodies (UltraSensitive S–P Kit; Maixin Biotechnology Company, Fuzhou, China) and color was developed with 3,30 diaminobenzidine hydrochloride (DAB) as chromogen, counterstained with Mayer’s hematoxylin, and mounted for evaluation using an Olympus (Japan) BX-51 microscope. In the negative control, the primary antibody was replaced by PBS. Slides were scored independently by two observers unaware of the identity of the samples, one of whom is a Professor of Pathology. Staining was scored for
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intensity (0, 1?, 2?, 3?) and percentage of membranous and cytoplasmic staining in malignant cells (1, 0–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%). The sum of intensity and percentage counts was used as the final score. We considered \8 as low-level expression, and C8 as highlevel expression. Follow-up study In this prospective study, patients were followed up for a period of more than two years. None of the patients received radiation or chemotherapy before surgery. Patients were given a physical examination every three months for one year post-operatively, and then every six months with brain CT examination. Disease progressionfree overall survival (OS), defined as the time from the date of surgery to the date of death or last contact if the patient was still alive, ranged from two to 30 months (median, 16 months). Among the 96 glioma patients in this study, complete follow-up was available for 70 patients. Twentyeight patients for whom recurrence was not observed were alive at the end of the follow-up period. In addition, 58 patients died owing to disease recurrence. Statistical analysis The difference between F10 promoter methylation status of normal brain tissues and glioma tissues was examined by use of the independent samples t test. The relationships between F10 methylation status, protein expression, and clinicopathological data were examined by use of the v2 test. OS curves were calculated by use of the Kaplan–Meier method. Results were regarded as significant when P was B0.05. All statistical analysis was performed with SPSS13.0 for Windows (SPSS, Chicago, IL, USA).
Results Clinical characteristics and histopathologic features The main clinical features of the patients, 42 women (43.8%) and 54 men (56.2%), are summarized in Table 1. The mean follow-up period from surgery was 492 days (range, 2–30 months). The frontal lobe was the most common location, and 31% of the patients (30/96) had frontal lobe tumors or both frontal and other lobes involved. Seizures were the most frequent presentations (34.3%); other, less frequent, symptoms included headache, vomiting, neurological deficit, and mental deterioration. All patients underwent operation. Extent of resection was determined by tumor location and size. Subtotal resection was defined when more than 95% of the tumor
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Table 1 Clinical parameters of patients Variable
Grade I
Grade II
Grade III
Grade IV
Total
No. of patients
14
42
28
12
96
Male
8
22
19
5
54
Female
6
20
9
7
42
\44
9
27
6
2
44
C44
5
15
22
10
52
Sex (n)
Age (years)
Follow-up (months) Median
23
17
15
7
Range
6–30
3–30
5–26
2–16
was removed, otherwise (i.e.\95%) resection was defined as partial. If conditions were suitable, subtotal resection was the first choice. Adjuvant therapy was not performed for 33 patients (34.4%); 21 (21.8%) received both radiotherapy and chemotherapy, 38 (39.6%) received radiotherapy only, and four (4.2%) received chemotherapy only. The median radiation dose was 54 Gy (range, 50–72 Gy) with no dose difference on the basis of sex or age. A combination regimen of temozolomide, carmustine, and vincristine was used most commonly. Forty-two tumors (43.8%) were classified as WHO grade II, 28 (29.2%) as WHO grade III, 14 (14.6%) as WHO grade I, and 12 (12.5%) as WHO grade IV (Table 1). In this study, histopathological grade correlated with survival (P = 0.000) with shorter overall and progression-free survival for grade IV tumors. F10 hypomethylation in glioma cells and tissues We examined whether this epigenetic alteration could be extrapolated to gliomas. We designed and validated BSP for F10 methylation within the region (Fig. 1a) which included 28 CpGs. The methylation status of the F10 promoter was determined by bisulfite sequencing for three glioma cell lines, 96 glioma samples, and 10 normal brain tissues. Tumor cell lines were significantly hypomethylated; DNA methylation decreases were SF126 38.6%, SF767 26.4%, and U251 24.3% (Fig. 1b; Table 2). CpG dinucleotides were heavily methylated in normal brain samples, and methylation decrease varied from 0 to 14.3% with a mean of 8.1% and a median of 9.3% (IQR 5, 10.7). In contrast, the methylation decrease observed in the glioma samples ranged from 7.6 to 35.7%, with a mean of 17.8% and a median of 16.3% (IQR 13.2, 22.3) (P = 0.000) (Fig. 1b). Methylation decrease levels [12.0% were regarded as hypomethylation. F10 was hypomethylated in 79 (82.3%) of 96 tumors.
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Fig. 1 a Schematic diagram of CpG dinucleotides within the F10 promoter. Nucleotide number is relative to the transcription start site of F10. The arrow indicates the region tested in BSP. b Bisulfite sequencing of the upstream regulatory region of F10 was performed for selected cell lines (SF126, SF767, and U251) and representative tissues (N normal brain tissue, T glioma sample). For each sample, at least five separate clones were sequenced and the results are shown here. Unmethylated CpG sites are shown as open circles whereas methylated CpG sites are indicated by closed circles. For each row of circles sequence results for an individual clone of the bisulfite-PCR product are given. The number of methylated CpGs divided by the total number of true CpGs analyzed is given as a percentage on the right of each bisulfite result
Correlation of F10 hypomethylation with clinicopathological characteristics For some tumors types hypomethylation of satellite DNA sequences has been significantly correlated with malignant potential, aggressive histological features, and worse prognosis [12]. Accordingly, we evaluated the methylation status of the F10 promoter in 96 brain tumor samples using BSP as described in the ‘‘Materials and methods’’ section. Most (79/96, 82.3%) of the samples were hypomethylated compared with the normal brain tissues. There was no statistically significant correlation between sex, age, histological grade, and F10 hypomethylation (Table 2).
J Neurooncol (2012) 107:479–485 Table 2 Correlation of F10 hypomethylation with clinicopathological characteristics
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Variable
Hypomethylation
Methylation
x2
P
0.167
1.907
0.012
0.911
0.557
0.345
Total (N = 96) Sex Male (54)
47
7
Female (42)
32
10
Age (years)a \44 (44)
36
8
C44 (52)
43
9
Low grade(I ? II)(56)
45
11
High grade(III ? IV)(40)
34
6
Grade
a
Median age is 44 years
Relationship between F10 hypomethylation and protein expression
Association of protein expression with clinicopathological data
Increased expression of FX protein in 75 of 96 glioma tissues was shown by immunohistochemical analysis. Comparison of methylation data with immunohistochemistry findings revealed high-level expression of FX protein in 67 (84.8%) of the 79 tumors harboring F10 hypomethylation. In comparison, reduced or loss of FX expression was observed in 9/17 (52.9%) of methylated tumors and moderate to high-level expression was observed in 8/17 (47.1%) (Fig. 2). There was a significant association between F10 promoter hypomethylation and its protein expression (P = 0.001, OR = 6.3, 95%CI = 2.0–19.5; Table 3).
There was no significant association between FX protein expression in tumors and sex, age, and tumor grade (P = 0.162, 0.239, 0.260; OR = 2.0, 0.6, 1.7; 95% CI = 0.8–5.3, 0.2–1.5, 0.7–4.6) (Table 3).
Fig. 2 Immunohistochemical analysis of FX. Paraffin-embedded tissue sections from gliomas and normal brain tissues were used for immunohistochemical analysis of FX protein with polyclonal antibody of FX, as described in the ‘‘Materials and methods’’ section, and
sections were counterstained with hematoxylin. The photomicrograph shows a normal brain tissue showing no expression of FX, b grade I, c grade II, d grade III, and e grade IV gliomas with immunopositivity. (a–e, original magnification 9200)
Clinical outcome of the patients Kaplan–Meier survival analysis was carried out to determine the prognostic potential of F10 hypomethylation and FX protein expression. Glioma patients with hypomethylated F10 promoter in tumors had shorter median OS (median OS = 15 months; 95%CI = 12–18) than patients
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Table 3 Association of protein expression with F10 hypomethylation and clinicopathological characteristics Variables
FX protein expression (score) \8
C8
12 9
67 8
x2
P
11.666
0.001
1.959
0.162
1.385
0.239
1.270
0.260
Total (N = 96) Methylation status Hypomethylation (79) Methylation (17) Sex Male (54)
9
45
12
30
\44 (44)
12
32
C44 (52)
9
43
Female (42) Age (years)
Grade Low grade(I ? II) (56)
10
46
High grade(III ? IV) (40)
11
29
without hypomethylated F10 promoter (median OS = 20, P = 0.025) (Fig. 3a). Similarly, the correlation between FX protein expression and OS was statistically significant. Markedly reduced OS was observed in glioma patients with FX overexpression (median OS = 16, P = 0.019; 95% Fig. 3 a Correlation between F10 methylation in tumor and OS of glioma patients. b Correlation between FX protein expression in the tumor and OS of glioma patients. c Comparison of OS of patients with high FX expression and patients with low-expression in the F10 hypomethylation group
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CI = 13–19) compared with patients with loss of its expression (Fig. 3b). In the F10 hypomethylation group, OS of patients with high F10 expression was compared with that of patients with low F10 expression. The results showed that the group with high expression of F10 had shorter median OS than the low-expression group (P = 0.018, Fig 3c). Because sensitivity to radiotherapy or chemotherapy is another aspect of prognosis, we analyzed the correlation between F10 hypomethylation and effect of radiotherapy and chemotherapy. In our study, 21 (21.8%) patients received both radiotherapy and chemotherapy, 38 (39.6%) received radiotherapy only, and four (4.2%) received chemotherapy only. Of 25 patients who received chemotherapy, eight patients were not sensitive, and the F10 methylation level for these was lower than for the sensitive group (P \ 0.01). Furthermore, in the radiotherapy group (59) the F10 methylation level of non-sensitive patients (13) was lower than that of sensitive ones (46) (P \ 0.01). This indicated that F10 hypomethylation might resistant to radiotherapy or chemotherapy.
Discussion At the epigenetic level, tumor specimens are most often characterized in terms of altered DNA methylation [4]. DNA
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methylation of promoter CpG islands has been recognized as an important mechanism for regulation of gene expression and transcriptional modification in mammals. Aberrations in DNA methylation patterns may have critical effects on tumor initiation and progression [13]. DNA hypomethylation of specific genes and repetitive DNA is observed in brain tumors [7, 14]. Although much work has been conducted on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes [15]. Although F10 is a coagulation-related gene, its expression in glioma, even in tumors, has not been mentioned. Until now, no information has been provided about regulation of the F10 gene in tumors. However, rather limited data are available on the relationship between the F10 gene and its precise mechanism of action on tumors, and their clinical effect on outcome for patients with glioma remains unclear. We hypothesized that F10 may be overexpressed in glioma and DNA hypomethylation may be its mechanism of up-regulation. In our methylation analysis, tumor cell lines were significantly hypomethylated with a DNA methylation decrease of SF126 38.6%, SF767 26.4%, and U251 24.3%. F10 was hypomethylated in 79 (82.3%) of 96 tumors, but no normal brain sample was hypomethylated. To investigate the epigenetic importance of F10 hypomethylation in gliomas, we performed immunohistochemistry which revealed that of 79 tumors harboring F10 hypomethylation, 67 (84.8%) had high-level expression of F10 protein, and there was a significant association between F10 promoter hypomethylation and its protein expression (P = 0.001). We then focused this investigation on evaluating the clinical effect of F10 in gliomas. The results showed there was no statistically remarkable correlation between sex, age, histological grade, and F10 hypomethylation. However, glioma patients with hypomethylated F10 promoter in tumors had shorter median OS. Otherwise, F10 protein expression in tumors was not remarkably associated with tumor grade, sex, and age. Outcome was poor for patients harboring high F10 expression. In addition, among 79 F10 hypomethylation patients, 67 harbored FX overexpression and the other did not. The different OS of FX highexpression and FX low-expression patients was then determined; the results showed that OS was shorter for the FX high-expression group than for the FX low-expression group. These observations indicated that glioma with F10 hypomethylation and higher protein expression might be a more biologically aggressive phenotype than those without it. These results showed that increased expression of FX in gliomas with a poor prognosis was at least partially because of hypomethylation of the F10 promoter region. F10 promoter hypomethylation was found to be an independent prognostic factor for OS. In addition, F10 hypomethylation may be correlated with response to
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radiotherapy and chemotherapy. It was shown that F10 methylation level was lower in patients with resistance to radiotherapy or chemotherapy. Nonetheless, on the basis of these observations and the results from subset analysis, it is reasonable to conclude that F10 promoter methylation status is an important prognostic biomarker in glioma. Acknowledgments This study was supported by grants from The 111 project (111-2-12), the National Science Foundation of China (81171932; 30901539; 30901718), the Program for New Century Excellent Talents in University (NCET-08-0562), and the Hunan Province Natural Sciences Foundations of China (11JJ1013).
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