Biomedicine & Pharmacotherapy 68 (2014) 209–212

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Original article

Decreased expression of ten-eleven translocation 1 protein is associated with some clinicopathological features in gastric cancer Bartosz Adam Frycz a, Dawid Murawa b, Maciej Borejsza-Wysocki c, Ryszard Marciniak c, Paweł Murawa b, Michał Drews c, Anna Kołodziejczak a, Katarzyna Tomela a, Paweł Piotr Jagodzin´ski a,* a

Department of Biochemistry and Molecular Biology, Poznan´ University of Medical Sciences, Poznan´, Poland First Department of Surgical Oncology and General Surgery, Wielkopolska Cancer Center, Poznan´, Poland c Department of General Surgery, Oncologic Gastroenterological and Plastic Surgery, Poznan´ University of Medical Sciences, Poznan´, Poland b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 November 2013 Accepted 31 December 2013

A decrease in ten-eleven translocation 1 (TET1) transcript and 5-Hydroxymethylcytosine (5hmC) levels has recently been demonstrated in primary gastric cancer (GC). However, little is known about TET1 protein levels in gastric tumoral and nontumoral tissue. Therefore, using reverse transcription, real-time quantitative polymerase chain reaction and western blotting analysis, we determined the TET1 transcript and protein levels in tumoral and nontumoral tissue from 38 patients with GC. We also assessed the association between the decrease in TET1 transcript and protein levels and some clinicopathological features in primary GC. We found significantly decreased levels of TET1 transcript (P = 0.0023) and protein (P = 0.00024) in primary tumoral tissues as compared to nontumoral tissues in patients with GC. Moreover, we also observed significantly lower amounts of TET1 transcript (P = 0.03) and protein (P = 0.00018) in tumoral tissues in patients aged > 60. We also found significant lowered TET1 protein levels in male patients (P = 0.0014), stomach (P = 0.044) and cardia (P = 0.013) tumor localization, T3 depth of invasion (P = 0.019), N1 (P = 0.012) and N3 lymph node metastasis (P = 0.013) and G3 histological grade (P = 0.0012). There were also significant decreases in TET1 transcript levels in female patients (P = 0.042), intestinal histological types (P = 0.0079) and T4 depth of invasion (P = 0.037). Our results demonstrated that a decrease in TET1 transcript and protein levels is associated with some clinicopathological features in GC. ß 2014 Published by Elsevier Masson SAS.

Keywords: Gastric cancer TET1 5-Hydroxymethylcytosine (5hmC)

1. Introduction Gastric cancer (GC) is one of the most common malignancies worldwide [1]. The pathogenesis of GC is linked to various genetic abnormalities including mutations and gene amplification [2]. Beyond these abnormalities, development of GC is also accompanied with epigenetics events, especially hypermethylation and hypomethylation of cytosine and guanine (CpG)-rich islands located in promoters of cancer-related genes [3,4]. The hypermethylation of CpG-rich islands may lead to either functional switch off or reduced expression of tumor-suppressor genes. Hypomethylation may result in transcriptional activation of protooncogenes, retrotransposons and genes involved in genomic

* Corresponding author. Department of Biochemistry and Molecular Biology, Poznan´ University of Medical Sciences, 6, S´wie˛cickiego St., 60-781 Poznan´, Poland. Tel.: +48 61 546519; fax: +48 61 546510. E-mail addresses: [email protected], [email protected] (P.P. Jagodzin´ski). 0753-3322/$ – see front matter ß 2014 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.12.011

instability and cell metastasis [5]. These DNA methylation abnormalities are due to deregulation of DNA methylation that maintains the machinery encompassing DNA methyltransferases and systems involved in DNA demethylation [6]. Although enzymatic pathways implicated in DNA methylation have been well characterized over recent years, the mechanisms of DNA demethylation – especially during carcinogenesis – remain unclear [7]. The significant role of ten-eleven translocation (TET) proteins in DNA demethylation during carcinogenesis was recently demonstrated. The mammalian TET family includes the TET1, TET2 and TET3 proteins, which display a high degree of homology within the C-terminal catalytic domain [8]. They are 2-oxoglutarate and Fe (II) dependent dioxygenases capable of sequential conversion of 5-methylcytosine (5mC) to 5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which leads to removal of a methyl group from 5mC [9,10]. The presence of TET proteins has been demonstrated in tissues in which the DNA demethylation process takes place [8,11]. The TET1 protein contains the CXXC zinc-finger motif, recognizing

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both 5mC and 5hmC in high CpG density DNA regions and unmethylated CpG dinucleotides close to start sites of transcription [12,13]. There are many reports demonstrating reduced levels of 5hmC in many types of cancers [14], including GC [15]. Furthermore, differences in the expression of all three TET genes have been demonstrated between cancerous and noncancerous tissues in carcinomas of the breast, liver, lung, pancreas and prostate. Decreased TET gene expression was associated with a reduction of the 5hmC level in cancer tissues [16]. It has recently been reported that decreased 5hmC levels are associated with some clinicopathological features in primary GC [17]. However, little is known about levels of TET1 protein in gastric tumoral and nontumoral tissue. Therefore, we studied the association between the TET1 transcript, protein levels and clinicopathological features in primary GC from 38 patients. 2. Materials and methods

2.2. Antibodies and reagents Rabbit polyclonal anti-TET1 antibody (Ab) was purchased from Biorbyt (Cambridge, UK). Rabbit polyclonal anti-glyceraldehyde-3phosphate (GAPDH) Ab (FL-335) and goat anti-rabbit horseradish peroxidase (HRP)-conjugated Ab were provided by Santa Cruz Biotechnology (Santa Cruz). 2.3. Reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR) analysis of TET1 transcript levels

2.1. Patient material Primary tumoral and nontumoral gastric tissues were collected between December 2012 and October 2013 from 38 patients who underwent total gastrectomy at First Department of Surgical Oncology and General Surgery Wielkopolska Cancer Center, Poznan´, Poland, or Department of General Surgery, Oncologic Gastroenterological and Plastic Surgery, Poznan´ University of Medical Sciences, Poland (Table 1). The procedures of the study were approved by the Local Ethical Committee of Poznan´ University of Medical Sciences. Written informed consent was obtained from all participating individuals. Histopathologically

Table 1 TET1 mRNA and protein level in nontumoral and tumoral tissues of patients with gastric cancer (GC) and their clinicopathological characteristics. Number of available data

P* mRNA

Protein

Age  60 > 60

6 29

P = 0.066 P = 0.03

P=1 P = 0.00018

Gender Male Female

21 15

P = 0.064 P = 0.042

P = 0.0014 P = 0.18

Site Stomach Cardia Body

24 6 1

P = 0.087 P = 0.17 –

P = 0.044 P = 0.013 –

Histological types Diffuse Intestinal Diffuse/intestinal

9 12 8

P = 0.48 P = 0.0079 P = 0.4

P = 0.54 P = 0.019 P = 0.083

Depth of invasion T1 T2 T3 T4

2 6 16 6

– P = 0.69 P = 0.073 P = 0.037

– P = 0.94 P = 0.019 P = 0.066

Lymph node metastasis N0 N1 N2 N3

10 5 8 6

P = 0.38 P = 0.4 P = 0.1 P = 0.2

P = 0.16 P = 0.012 P = 0.96 P = 0.013

Malignancy grade G1 G2 G3

1 7 25

– P = 0.074 P = 0.056

– P = 0.44 P = 0.0012

Variables

unchanged gastric mucosa situated at least 10–20 cm away from the tumoral lesions was obtained from the same patients. A part of the samples was immediately snap-frozen in liquid nitrogen and stored at 80 C until RNA and protein isolation. The second part was fixed in 10% formalin then cleared, dehydrated and paraffinembedded for histologic examination. Histopathological assessment encompassing depth of invasion, histological type, lymph node metastasis and malignancy grade was performed by an experienced pathologist.

Statistically significant results are shown as bold. *P < 0.05 was considered as statistically significant. The P value was evaluated by U-Mann-Whitney test.

The total RNA from tumoral and nontumoral primary tissues of 38 patients with GC was isolated according to the method of Chomczyn´ski and Sacchi [18]. RNA samples were treated with DNase I, quantified, and reverse-transcribed into cDNA. RQ-PCR was carried out in a Light Cycler real-time PCR detection system from Roche Diagnostics GmbH, (Mannheim, Germany) using SYBR1 Green I as detection dye. The transcript levels in the patient tissues were expressed as multiplicity of these cDNA concentrations in the calibrator. The calibrator was prepared as a cDNA mix from all of the patient samples and successive dilutions were used to create a standard curve as described in Relative Quantification Manual Roche Diagnostics GmbH (Mannheim, Germany). For amplification, 1 mL of cDNA solution was added to 9 mL of qPCR Master Mix EURx company (Gdansk, Poland) and the following primer sequences: TET1 forward: 5’-CCATCTGTTGTTGTGCCTC-3’, TET1 reverse: 5’-CAGTATGTGGGTTCAATTCC-3’. The quantity of TET1 transcripts in each sample was standardized by the geometric mean of porphobilinogen deaminase (PBGD) and human mitochondrial ribosomal protein L19 (hMRPL19) cDNA levels [19]. 2.4. Western blotting analysis To determine the TET1 protein levels, primary tissues from patients with GC were treated with lysis RIPA buffer, and 30 mg of protein were resuspended in sample buffer and separated on 8% Tris-glycine gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gel proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% milk in Tris/HCl saline/Tween buffer. Immunodetection of bands was performed with rabbit polyclonal anti-TET1 Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. To ensure equal protein loading of the lanes, the membrane was re-stripped and incubated with rabbit polyclonal anti-GAPDH Ab and goat anti-rabbit HRP-conjugated Ab. Bands were revealed using ECL kit and Hyperfilm ECL Amersham (Piscataway, NJ). The amount of TET1 protein was presented as the TET1-to-GAPDH band optical density ratio. 2.5. Statistical analysis The normality of the observed patient data distribution was assessed by Shapiro-Wilk test, followed by unpaired, two-tailed ttest or Mann-Whitney test to consider statistically significant differences of compared mean values. Statistical analysis was performed with Statistica 10 software.

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3. Results and discussion Studies conducted in mouse models demonstrated the essential role of TET1 in maintaining decreased 5hmC levels at the promoters of embryonic stem (ES) cell specific genes [20–22]. Both TET1 and 5hmC levels have been abundant in murine ES cells, in the inner cell mass of the murine blastocyst, and in cells of the early murine epiblast [22–24]. Recent studies suggest that there might be several pathways or mechanisms by which TET proteins, oxidizing 5mC to 5hmC, control DNA methylation and gene transcription [25]. In the passive mechanism of DNA demethylation, 5hmC cannot be recognized by DNA methyltransferase, maintaining the DNA methylation patterns [26]. Moreover, TET proteins can participate in passive DNA demethylation mechanisms by TETs binding to unmethylated CpG dinucleotides in the promoter region and inhibition of DNA methylation [8,13,27,28]. There are several suggested active mechanism of DNA demethylation, including successive oxidation of 5mC leading to the removal a methyl group. Other mechanisms include deamination of 5hmC by activation-induced deaminase to 5-hydroxymethyluracil, which is subsequently recognized by base excision repair enzymes (BER) leading to DNA demethylation [29]. Another possible active demethylation mechanism encompasses conversion of 5fC to 5caC, which can be subsequently cut off by thymine DNA glycosylase and corrected by the BER system [30]. The role of abnormalities in the machinery of DNA demethylation has recently being considered in the development of various cancers [31]. In our study we found significantly decreased levels of TET1 transcript (P = 0.0023) and protein (P = 0.00024) in primary tumoral as compared to nontumoral tissues from patients with GC (Fig. 1A, B, C). Moreover, we observed significantly lower amounts of TET1 transcript (P = 0.03) and protein (P = 0.00018) in tumoral tissues in patients aged > 60 (Table 1). We also found a significant association between lower TET1 protein levels in male patients (P = 0.0014), stomach (P = 0.044) and cardia (P = 0.013) tumor localization, T3 depth of invasion (P = 0.019), N1 (P = 0.012) and N3 lymph node metastasis (P = 0.013) and G3 histological grade (P = 0.0012). We also observed a significant decrease in TET1 transcript in female patients (P = 0.042), intestinal histological types (P = 0.0079) and T4 depth of invasion (P = 0.037). Recently, the study conducted by Yang et al. [17] demonstrated reduced levels of TET1 transcript associated with decreased 5hmC levels in primary gastric cancerous tissue. Furthermore, they demonstrated that decreased 5hmC correlated with tumor size, Bormman type, tumor invasion, TNM stage, lymph node metastasis and cancer-related death [17]. The changes in levels of TET1 transcript [15,32,33] and protein [33–35] associated with 5hmC distribution in genomic DNA have also been demonstrated in other malignancies. Down-regulation of TET1 transcript in cancerous tissue as compared to matched normal tissue has been proposed as a main mechanism in loss of 5hmC in genomic DNA during colonic carcinogenesis [15]. The up-regulation of TET1 transcript leads to a global increase of the 5hmC level in mixed lineage leukemia gene rearranged in acute myeloid leukemia cells [36]. Moreover, it has been demonstrated that TET1 inhibits DNA methylation in genes encoding tissue inhibitors of the metalloproteinase (TIMP) family proteins in normal as well as in breast and prostate cancer cells in vitro. In this study, a decreased TET1 transcript level linked to TIMPs down-regulation was associated with increased cell invasion, tumor growth, and metastasis in murine xenograft models of prostate cancer, as well as poor survival rates in patients with breast cancer [33]. In addition to these findings, changes in TETs gene expression and 5hmC levels in genomic DNA in tumoral tissue have also been correlated with some clinicopathological features of cancers, tumor recurrence and survival of patients [33,34,37,38]. The loss of TET1 expression and 5hmC in genomic

Fig. 1. TET1 transcript (A) and protein (B) levels and representative picture of western blot (C) in primary nontumoral (N) and tumoral (T) tissues from patients with GC. The nontumoral (*) and tumoral (*) tissues from 38 patients with GC were used for RNA and protein isolation. Total RNA was reverse-transcribed, and cDNAs were studied by RT-PCR analysis. The TET1 mRNA levels were adjusted by the geometric mean of PBGD and hMRPL19 cDNA levels. The amounts of TET1 mRNA were expressed as multiples of these cDNA copies in the calibrator. Proteins were separated by 8% SDS-PAGE and transferred to a membrane that was then immunoblotted with rabbit polyclonal anti-TET1 Ab and incubated with goat antirabbit HRP-conjugated Ab. After stripping, the membrane was reblotted with rabbit polyclonal anti-GAPDH Ab and goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The amount of western blot-detected TET1 proteins was presented as the TET1-to-GAPDH band optical density ratio. The P value was evaluated by Mann-Whitney test.

DNA in human hepatocellular carcinoma correlated with tumor size, alpha fetoprotein levels and poor overall survival [34]. Moreover, that study’s authors observed that lower 5hmC levels in nontumoral tissues corresponded to tumor recurrence in the first year after surgical resection [34]. Decreased levels of isocitrate dehydrogenase 2 and all three TET mRNA have been associated with reduced 5hmC levels in genomic DNA and a poor prognostic factor in patients with melanoma [32]. In our study we did not find differences in TET2 transcript and protein levels between cancerous and noncancerous tissues (data not shown). Moreover, we did not observe a correlation between TET2 transcript and protein and clinicopathological features in GC (data not shown). Our observations are partially consistent with previous studies that have also demonstrated no differences in TET2 and transcript levels between lung [33], liver [34], brain [35] and gastric tumoral and nontumoral tissues [17]. Moreover, TET2 gene and protein expression was not correlated to the level of 5hmC in liver cancer [34] and GC [17]. However, the TET2 gene displayed a high frequency of mutation in myeloid malignancies [39].

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4. Conclusion We observed that decreased levels of TET1 transcript and protein in primary cancerous tissues correlate with some clinicopathological features in GC. Our studies were carried out in a relative small sample size of patients. Therefore, further studies in a larger group of patients are required to evaluate the correlation between TET1 transcript and protein levels in cancerous tissues and clinical characteristic of GC.

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