High expression of DNA methyltransferases in primary human medulloblastoma

Introduction

Medulloblastoma (MB) is the most frequent malignant brain tumor in childhood, with various histological appearances. There are non-overlapping histological (classic, desmoplastic, extensive nodular, large cell/anaplastic) and molecular classifications (SHH, WNT, Group 3 and 4) for medulloblastoma [3,6,11,23,25,27,34]. Despite multimodal therapies, the 70-75% long-term survival rate has not been exceeded yet in high-risk patients [13,29,34]. A more precise classification of patients would help to develop a more effective treatment strategy and reduce side effects [3,11,23].
There is emerging evidence that epigenetic changes contribute to carcinogenesis. The most important mechanisms of epigenetic alteration are DNA methylation, histone modification and microRNA regulation. DNA methyltransferases (DNMTs) are enzymes transferring methyl groups to cytosine in CpG dinucleotides, resulting in gene silencing. The DNMT family has three active members: DNMT1, DNMT3A and DNMT3B. DNMT3A and DNMT3B are regarded as de novo methyltransferases, whilst DNMT1 reacts on hemimethylated DNA, copying the methylation pattern during replication. Generally, tumor cells are characterized by global hypomethylation and hypermethylation of certain gene promoters. Therefore, repetitive elements are hypomethylated and specific tumor suppressor genes are repressed due to hypermethylation. These may contribute to chromosomal instability and abnormal gene silencing that could promote tumor-initializing steps [2,9,14,28,38]. Promoter hypermethylation of tumor suppressor genes and DNA repair genes has been observed in a wide variety of tumors, such as colorectal, breast cancer, glioma and medulloblastoma [19,22,36]. Overexpression of DNMTs was detected at the mRNA or protein level. Increased DNMT1 expression was described in gastric cancers, along with DNMT3A in breast and pancreatic cancer, or along with DNMT3B in glioma [8,16,30,38,39]. Although there is emerging evidence that epigenetic events play a remarkable role in the development of adult cancers, our knowledge about their role in pediatric brain tumors is still very limited.
DNA methylation studies in medulloblastoma have focused on the role of tumor suppressor genes in the development of brain tumors, and shed light on contradictory results in the majority of genes, except for RASSF1A [37]. Recent studies indicate that the DNA methylation profile can be used for MB subgrouping and disease risk assessment [18,35].
The aims of our study are to characterize the expression of DNMTs in human medulloblastoma samples and their potential prognostic role in survival of patients. Possible correlations of DNMT proteins and clinicopathological features are also analyzed.

Material and methods

Medulloblastoma tissue samples

Formalin-fixed, paraffin-embedded MB tumor samples were collected from the National Institute of Neurosciences (Budapest, Hungary) and 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University (Budapest, Hungary). Forty-four primary medulloblastoma samples removed surgically between 2004 and 2010 and related clinical data of patients derived from the Hungarian Pediatric Cancer Registry were used. The study was approved by the Ethics Committee of the Institutional Ethical Review Board of Sem­melweis University (TUKEB No. 30/2015).

Tissue microarray (TMA) construction and immunohistochemistry

Hematoxylin and eosin (HE) stained sections were reviewed by a neuropathologist to select representative areas for producing TMA. TMA blocks were created by a computer-controlled TMA Master (3DHISTECH, Budapest, Hungary) instrument. Depending on sample quality one or two cores of representative areas with a diameter of 2 mm were taken from each tumor sample. From the TMA blocks 4 µm sections were created. Sections were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked. Heat-induced antigen retrieval was performed with Tris-EDTA buffer (pH = 9.0) for 45 min in a microwave oven. Sections were incubated overnight using anti-DNMT1 antibody (dilution 1 : 200; Ab19905, Abcam, Cambridge, UK), anti-DNMT3A antibody (dilution 1 : 600; Ab13888, Abcam, Cambridge, UK) and anti-DNMT3B antibody (dilution 1 : 200; Ab13604, Abcam, Cambridge, UK) diluted in 5% BSA containing Tris-buffered saline (TBS; pH = 7.4). Antigen development was performed by Novolink Polymer Detection System (Novocastra, Wetzlar, Germany) following the manufacturer’s instructions. Reactions were visualized by 3,3’-diaminobenzidine (DAB) (DAKO, Denmark) as a substrate, and then slides were counterstained by hematoxylin. Tonsil for DNMT1, and kidney for DNMT3A and DNMT3B were used as positive tissue controls.
For determination of the WNT subgroup anti--catenin antibody (dilution 1 : 150; M-3539, DAKO, Denmark) and of the SHH subgroup anti-secreted frizzled-related protein 1 (SFRP1) antibody (dilution 1 : 1500; ab-4193, Abcam, Cambridge, UK), characteristic proteins of these pathways, were applied. Immunohistochemical reactions were executed as described above, but antigen retrieval was performed in TE buffer boiled in a pressure cooker for 20 minutes.

Evaluation of immunostaining

Slides were digitalized by a Pannoramic Scan instrument and examined by Pannoramic Viewer software (3DHISTECH, Budapest, Hungary). To evaluate DNMT expression only nuclear staining was considered. Intensity of staining and rate of positive cells were calculated from 1,000 cells in a representative area of each sample. According to the intensity of staining, four values were created: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The proportion of positive cells was ranked in five groups: 0 (negative), 1 (1-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%). The products of the two values (0-12) were divided into two groups: 0-3 regarded as no/weak expression and 4-12 regarded as moderate/strong expression. Samples were classified into WNT or SHH subgroups when the proportion of positive cells with nuclear -catenin or negative nuclear -catenin and positive SFRP1 staining was above 10% [7,27].

Statistical analysis

All statistical analysis was performed using Statistica software (StatSoft Inc., Tulsa, OK, USA). Survival of patients was calculated by the Kaplan-Meier method. Correlation of expression of DNMTs and survival was examined by the log-rank test. Correlation of expression of each DNMT was analyzed by the Spearman rank order test. Correlation of expression of DNMTs and age, gender, histological subtype or molecular subgroups was examined by the Mann-Whitney U-test and Fisher’s exact test, when appropriate. Results were considered statistically significant when the p value was less than 0.05.

Results

Patients’ data

Demographic characteristics of the studied cohort are summarized in Table I. Median age of 44 patients was 8.5 years (range 1.1-28.7 years). The proportion of male patients was 55%. Histologically, 84.1%, 11.4% and 4.5% of patients showed classic, desmoplastic and large cell/anaplastic (L/A) type of medulloblastoma, respectively. Survival data were available in 40 patients (90.9%). Median follow-up time was 5.6 years (range 0-9.7 years). Patients were treated according to the Hungarian MBL2004/2008 schedules [15]. There is no standard treatment for adults with medulloblastoma in Hungary. Patients are treated according to the individual decision of each treating physician, and these treatment details are poorly accessible in Hungary. Therefore, adult patients were omitted from the survival analysis.

Expression of DNMTs in human medulloblastoma

Elevated (moderate/strong) expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of patients, respectively (Table I). Representative stains are shown in Figure 1. The relationship of histology and DNMT expression was evaluated only in the classic and desmoplastic type. Moderate/strong expression of DNMT1 was detected in 62.2% of classic and 60.0% of desmoplastic, of DNMT3A in 70.3% of classic and 60.0% of desmoplastic, and of DNMT3B in 70.3% of classic and 80.0% of desmoplastic samples. We could analyze only two samples of L/A subtype. Both of them showed moderate/strong expression for DNMT1 and DNMT3B, but only one for DNMT3A.

Correlation between DNMT expression and patients’ data

There was no correlation between the expression of DNMT1, DNMT3A and DNMT3B and age at disease onset or gender or histological subtype (Table II). Kaplan-Meier curves, based on different expression of DNMTs, did not show a significant difference in overall survival (Fig. 2). None of the DNMTs could be used as a prognostic marker for medulloblastoma in our cohort. DNMT1 and DNMT3A (p = 0.08), or DNMT1 and DNMT3B (p = 0.17) or DNMT3A and DNMT3B (p = 0.69) did not show a co-expression pattern. Expression of nuclear -catenin was shown only in one patient (2.3%) with weak staining. There were 12 (27.3%) patients belonging to SHH subgroup according to their cytoplasmic, membranous or secreted SFRP1 expression and lack of nuclear -catenin (Table I). Expression of DNMT1 in the SHH subgroup compared to the non-WNT/non-SHH subgroup of patients was significantly higher (p = 0.02), whereas expression of DNMT3A and DNMT3B did not differ significantly between different groups (p = 0.78 and p = 0.17, respectively, Table II).

Discussion

In recent years a new prognostic classification of medulloblastoma was introduced by molecular findings, whilst there are still several twists in this classification system. It is still motivating to find further prognostic markers [3,6,11,23,25,27,34]. Nowadays, an exact diagnosis of pediatric brain tumors can be established based on the methylation profile of different tumor types [18,21,35]. DNMTs are responsible for DNA methylation, an important epigenetic regulation process, which contributes to development of cancer. DNMT1 is responsible for maintenance of methylation patterns during DNA replication. DNMT3A and DNMT3B are regarded as de novo methyl-transferases and abundantly expressed during embryonic development [2,9,14,19,22,28,38].
In our study, expression of three members of the DNMT family (DNMT1, DNMT3A, DNMT3B) in me­dulloblastoma samples and their possible effect on disease outcome were examined. During the development and progression of MB, the biological role of DNMTs, crucial regulators of the methylation pattern, is not completely understood yet. In our study, the correlation of expression of these enzymes at the protein level and clinical data of MB patients were investigated.
In our experiments elevated expression of all DNMTs was observed in most of the MB samples. Correlation of expression of DNMTs and histological subtype of MB, age at disease onset or gender was not proven in our experience. Increased DNMT1 expression indicates worse outcomes in several types of cancers, such as gastric cancer, hepatocellular carcinomas and pancreatic ductal adenocarcinoma [10,26,33]. In contrast, our analysis showed no difference in disease outcome when comparing patients with no/weak and moderate/strong expression of three DNMTs, so DNMTs are not predictive markers for medulloblastoma survival. The different result may be due to the different biological nature of medulloblastoma and above-mentioned tumor types. While there are some common dysregulated signaling pathways (WNT, SHH, Notch) in medulloblastoma and in these other cancer types, differences could also be observed in regulation of the following pathways: in gastric cancer altered EGFR, ErbB, mTOR, VEGF, HGF/MET pathways, etc.; in hepatocellular carcinoma altered Ras/Raf/MAPK, mTOR, etc.; and in pancreatic ductal adenocarcinoma FGF, Notch, TGF-, EGF and retinoid signaling [1,31,32].
The SHH subgroup of MB is characterized by the expression of SFRP1 and lack of nuclear -catenin [17,27]. Our results indicate that SHH pathway activation is associated with elevated DNMT1 expression, which could be a consequence of upregulation of DNMT1 by SHH in SHH-associated medulloblastomas. Similar observations were made in pancreatic cancer and in myelodysplastic syndrome (MDS) [16,40]. DNMT1 and DNMT3A could be regulated by Gli1, an effector of the SHH signaling pathway. The SHH pathway was inhibited in pancreatic cancer and MDS cell lines, resulting in decreased DNMT1 expression, whereas induction of the SHH pathway resulted in higher DNMT1 expression [16,40]. In contrast, DNMT1 inhibition resulted in increased expression of SFRP1 in MB cell lines [20]. One explanation could be that investigated MB cell lines do not belong to the SHH subgroup, since they showed lower levels of SFRP1 compared to the SHH subgroup. Among non-WNT/non-SHH patients, other mechanisms could result in DNMT1 activation. Further studies are needed to clarify the relationship between regulation of DNA methylation and SHH signaling pathway.
Theoretically, inhibition of DNA methylation is a therapeutic opportunity by reversing gene silencing [9,12,14,38]. An in vitro experiment showed that the DNMT inhibitor 5-aza-2’-deoxycytidine (decitabine) reactivates the tumor suppressor PTCH1 gene in the MB cell line [4]. A combination of DNMT and histone deacetylase (HDAC) inhibitors was effective to prevent MB development in Ptch knockout mice, while in advanced stage tumors this therapy was less efficient [5]. Epigenetic modulators combined with multi-kinase inhibitor could enhance apoptosis in vitro in medulloblastoma cells [24].
In conclusion, this is the first study to analyze DNMT expression in medulloblastoma in terms of expression level and correlation with clinical data. Elevated expression of DNMTs in human medulloblastoma, and association of DNMT1 and SHH subgroups were observed, without an effect on survival. Based on increased expression of DNMTs in me­dulloblastoma, their inhibition has potential for further investigation to optimize therapy of medullobla­stoma.

Acknowledgments

We thank Edit Parsch and Zita Bratu for excellent technical assistance, Miklós Garami for assistance in performing experiments and Balázs Hauser for help in preparation of the manuscript.

Disclosure

The authors report no conflict of interest.

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