RESEARCH ARTICLE Increased Hypermethylation of Glutathione S-Transferase P1, DNA-Binding Protein Inhibitor, Death Associated Protein Kinase and Paired Box Protein-5 Genes in Triple-Negative Breast Cancer Saudi Females

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with higher metastatic rate and both local and systemic recurrence compared to non-TNBC. The generation of reactive oxygen species (ROS) secondary to oxidative stress is associated with DNA damage, chromosomal degradation and alterations of both hypermethylation and hypomethylation of DNA. This study concerns differential methylation of promoter regions in specific groups of genes in TNBC and non-TNBC Saudi females in an effort to understand whether epigenetic events might be involved in breast carcinogenesis, and whether they might be used as markers for Saudi BCs. Methylation of glutathione S-transferase P1 (GSTP1), T-cadherin (CDH13), Paired box protein 5 (PAX5), death associated protein kinase (DAPK), twist-related protein (TWIST), DNA-binding protein inhibitor (ID4), High In Normal-1 (HIN-1), cyclin-dependent kinase inhibitor 2A (p16), cyclin D2 and retinoic acid receptor-β (RARβ1) genes was analyzed by methylation specific polymerase chain reaction (MSP) in 200 archival formalin-fixed paraffin embedded BC tissues divided into 3 groups; benign breast tissues (20), TNBC (80) and non-TNBC (100). The relationships between methylation status, and clinical and pathological characteristics of patients and tumors were assessed. Higher frequencies of GSTP1, ID4, TWIST, DAPK, PAX5 and HIN-1 hypermethylation were found in TNBC than in non-TNBC. Hypermethylation of GSTP1, CDH13, ID4, DAPK, HIN-1 and PAX5 increased with tumor grade increasing. Other statistically significant correlations were identified with studied genes. Data from this study suggest that increased hypermethylation of GSTP1, ID4, TWIST, DAPK, PAX5 and HIN-1 genes in TNBC than in non-TNBC can act as useful biomarker for BCs in the Saudi population. The higher frequency of specific hypermethylated genes paralleling tumor grade, size and lymph node involvement suggests contributions to breast cancer initiation and progression.


Introduction
Breast cancer (BC) is the most common malignancy among females worldwide and represents 22% of all newly diagnosed female cancers annually in Saudi Arabia with 60-80% of the patients are diagnosed at advanced stage (Al Tamimi et al., 2010;Aboserea et al., 2011). Triple Negative BC (TNBC) is a subtype of BC is characterized by the absence of hormone receptors expression of progesterone (PR), estrogen (ER) and human epidermal growth factor receptor-2 (HER-2) and represents 10-17% of all BC patients . It is well documented that tumor development requires phenotypic changes associated with initiation, promotion and progression phases carcinogenesis (Vincent and Gatenby, 2008). Genes in each of these phases acquire alterations in their transcriptional activity that are associated either with hypermethylation-induced transcriptional repression such as tumor suppressor genes or hypomethylation-induced activation in oncogenes (Panayiotidis, 2014). Comprehensive gene analyses on BC tissues discovered the expression profiles of genes and resulted in identification of mRNAs that are up-and down-regulated in breast carcinomas compared to normal breast tissue (Gypas et al., 2011;Wu et al., 2012). The DNA gene promoter hypermethylation is the major mode of tumor-specific up-down-regulation in number of genes (Kaczmarczyk et al., 2012;Tuononen et al., 2012).
These genes helped to explain the molecular detection and pathogenesis of BC as even small amounts of methylated sequences are readily detecTable. As little information is available about DNA methylation status in TNBC, therefore this study investigates the differential methylation of the promoter region in specific group of genes in TNBC and non-TNBC Saudi's females using MSP in an effort to understand the contribution of this epigenetic event in breast carcinogenesis, and whether it can be used as marker. The selected genes, those play a role in development and progression of BC, fall into different groups: genes commonly methylated in BC (including cyclin D2, CDH13 and RARβ1, P16, HIN-1), genes associated with transcription factors (such as TWIST, PAX5 and ID4), genes associated with DNA apoptosis such as (DAPK) and gene involved in detoxification of xenobiotics (such as GSTP1).

Materials and Methods
This study was conducted in compliance with Helsinki Declaration and has been approved by the Ethical Committee, College of Medicine, King Saud University. The present study based on 200 Saudi females living in the province of Riyadh, Kingdom of Saudi Arabia, with primary invasive breast cancer whom had undergone surgery from January 2009 to January 2011 (retrospective analysis). Two hundred formalin-fixed paraffin-embedded (FFPE) breast carcinoma tissues were collected from Pathology Department, College of Medicine, King Saud University (135 cases) and Pathology Department, Al-Shemissy Hospital (65 cases), Riyadh, Saudi Arabia. Inclusion criterion was archived primary breast tumors (T-stage 1-3 invasive ductal carcinoma of NOS type) assessed by immunohistochemistry for expression of ER, PR and HER-2 at the time of diagnosis. Whole tumor sections were examined to carefully review the histological characteristics of each tissue specimen.

Study design, demographic and clinical characteristics
A total of 200 FFPE samples were classified into three separate groups. Group 1 (Control): included 20 FFPE benign breast tissues and served as control. Group 2 (non-TNBC): included 100 FFPE samples from non-TNBC patients. Group 3 (TNBC) included 80 FFPE samples from TNBC patients. In non-TNBC patients, the mean age at diagnosis was 48 years (SE 1.12; range 24 to 91 years). Twenty-two cases were less than 40 years, 46 cases were 40-50 years and 32 cases were above 50 years. All Non-TNBC cases had invasive ductal carcinoma with 65 cases had grade III, 29 had grade II and 6 had grade I. Moreover, more than 50 % of non-TNBC cases (53) were positive for ER, PR and HER-2-neu, 19 cases were ER and PR negative and HER-2-neu positive, 15 cases were ER and PR positive and HER-2-neu negative and 13 cases were ER and HER-2-neu positive and PR negative. On the other hand, in TNBC patients, the mean age at diagnosis was 42.5 years (SE 1.2; range 28 to 70 years). A total of 36 cases were less than 40 years, 28 cases were 40-50 years and 16 cases were above 50 years. Regarding the histoloical categories, all TNBC cases had invasive ductal carcinoma with 35 cases had grade III, 36 had grade II and 9 had grad I. Moreover, all TNBC cases (80) were negative for ER, PR and HER-2-neu. These selected characteristics of the patients showed that there is no significant difference between TNBC and Non-TNBC cases regarding the mean age, histological categories and histological grade of the tumors.

DNA extraction and bisulfate treatment
All the FFPE samples were thin sectioned at 8 µm thicknesses using Leica Microtome (Manual Rotary Microtome RM2235). Tissue sections were floated in a DEPC-treated water bath then picked up on clean glass slides and allowed to air dry at 4°C, after which they were stored at -20°C until used. The composition of the unstained slides from each archival FFPE tissue block studied was confirmed by histopathologic examination of surrounding hematoxylin and eosin (H and E)-stained sections. For each tumor, the lesion was identified on an initial H and E-stained section and confirmed to remain on a serial H and E section taken following preparation of unstained sections for micro-dissection and nucleic acid extraction.
Hence, the lesion was documented to be present on stained sections taken before and after preparation of the analyzed unstained sections. For DNA extraction, two 8µm tissue sections were examined and selected areas of tumor tissues were removed from the slides using scalpel and placed directly into sterile 2ml Eppendorf tubes. DNA was extracted after micro-dissection using Recover All total Nucleic Acid Isolation Kit (Ambion, Life Technologies, USA) following the manufacturer instructions. Tissue sections were deparaffinized followed by digestion using buffer and proteases. Nucleic acids were isolated with additive/ethanol mixture followed by transfer to the column and elution. Quantity and quality of extracted DNA as characterized using a UV spectrophotometer (NanoDrop 8000, thermo scientific, USA).

Sodium bisulfite treatment of DNA
Tissue DNA was treated with sodium bisulfate using DOI:http://dx.doi.org/10.7314/APJCP.2015.16.2.541 Increased DNA Hypermethylation in Triple-negative Breast Cancer EpiTect Bisulfite Kits (QIAGEN, Germany) according to the manufactures' instructions. This process converts nonmethylated cytosine residues to uracil, whereas methylated cytosines remain unchanged. All solutions were prepared fresh. Briefly 50 µl DNA (1-2 μg) extract was incubated with 140 µl of bisulfite reaction components at room temperature for 5min followed by denaturation at 95°C for 5min, 60°C for 25 min, 95°C for 5min, 60°C for 85 min, 95°C for 5 min and finally 60°C for 175 min. BL buffer containing 10 μg/ml carrier RNA was mix with bisulfite converted DNA and transfer to EpiTect spin columns. After washing, 20 μl of Elution Buffer was added onto the center of each membrane then the DNA was aliquoted and stored until used at -80°C.

Methylation-specific polymerase chain reaction (MSP) using real time PCR
CpG islands of CDH13, GSTP1, ID4, p16, TWIST, Cyclin D2, PAX5, DAPK, HIN-1 and RARβ1 genes were examined by MSP (TAKARA, BIO INC, Japan). Forward and reverse primers were synthesized (Metabion, Germany) corresponding to the predicted sequence of methylated or unmethylated genomic DNA after sodium bisulfate treatment (Table 1). For reaction, 1 µl sodium bisulfitetreated DNA was added to 24 µl reaction buffer containing 0.3 µM of forward and reverse primers specific to unmethylated and methylated DNA sequences, 1.2 µl of MSP enzyme, 0.5µl of 100X SYBRGreen1 and 1 µl of 50X of ROX reference Dye. The cycling conditions were 95°C for 5 min, followed by 45 cycles of 98°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec. Universally methylated DNA and unmethylated DNA (CHEMICON International, Temecula, CA, USA) were used as controls for MSP. Following amplification melting curve analysis was performed to identify the presence of primer dimers and analyzed the reaction specificity. MSP products were separated electrophoretically on 3% agarose. Single band of methylated and/or unmethylated genes were visualized by photo-documentation system (syngene bio imaging, USA) (Figure1).

Statistically analysis
The exact chi-square test (SPSS, version 15.0) was used to analyze the association between methylation frequencies of all genes and to compare the tumor characteristics (e.g., age, tumor grade, tumor size) in breast cancer groups. P values <0.05 were considered to be statistically significant.

Table 1. Primers sequence of Methylated (M) and Unmethylated (U) Genes Used for Methylation Specific Polymerase Chain Reaction
In TNBC, DAPK gene hypermethylation was significantly increased with tumor size from 77.8% in <2 cm to 84.6% in >2 cm compared to 33.3% and 46.8% in Non-TNBC respectively. In TNBC, DAPK gene hypermethylation was significantly associated also with grade increasing 77.8% in grade-I, 69.4% in grade-II and 85.7% in grader-III cases compared to 33.3%, 44.8% and 43.1% respectively in non-TNBC. According to LN status in TNBC, DAPK gene was significant increase from 74.3% in LN negative to 80% in LN positive compared to 41.2% and 46.9% respectively in non-TNBC (Table 4).
ID4 gene hypermethylation was significantly increased with increasing tumor size 84.6% in >2 cm compared to 72.2% in <2 cm in TNBC compared to 46.8% and 33.3% respectively in non-TNBC (Table 3). There was significant increase in association with LN status in TNBC 77.8% in LN positive and 68.6% in LN negative compared to 49.9% and 41.2% respectively in non-TNBC. With regard to tumor grade, a significant hypermethylation of ID4 gene (66.7% and 80%) was observed in grade II and III in TNBC compared to Non-TNBC. GSTP1 gene hypermethylation in TNBC was significantly associated with tumor grade 66.7% in grade-I, 75% in grade-II and 71.4% in grade-III compared to 16.7%, 27.6 and 33.8% respectively in non-TNBC (Table 2). In relation to LN status, no significant changes were observed in both groups. Concerning tumor size, GSTP1 gene hypermethylation was significantly increased with tumor size in TNBC compared to Non-TNBC.
Cylcin D2 hypermethylation was significantly higher   in tumor grade I (83%) in non-TNBC compared to 33.3% in TNBC groups. In both groups, there was no association between hypermethylation of cyclin D2 and tumor size. According to LN status, in non-TNBC was 21.9% in LN positive compared to 38.2% in LN negative while in TNBC no association was observed (Table 2).
RARβ1 gene hypermethylation was insignificantly increased with tumor size, in TNBC was 34.6% in tumor size >2 cm and 16.7% in those <2 cm compared to 24.2% and 37.5% respectively in non-TNBC. There was a decrease in this gene associated with LN status by 28.9% in LN positive and 20% in LN negative in TNBC compared to 21.9% and 27.9% respectively in non-TNBC (Table 2).
PAX5 gene hypermethylation was decreased with tumor size <2 cm and >2 cm from 61.1% to 53.8% in TNBC and increased in non-TNBC from 41.7% to 58.1% respectively. In TNBC, hypermethylation of PAX5 was associated with grade increasing 55.6% in grade-I, 52.8% in grade-II and 60% in grade-III compared to 66.7%, 62.1% and 47.7% respectively in non-TNBC. In relation to LN status, in TNBC hypermethylation was increased from 51.4% in LN negative to 60% in positive LN and decreased from 59.4% to 50% in non-TNBC, but this differences were statistically insignificant (Table 3).
TWIST gene hypermethylation was observed with high frequency in grade-I 55.6% in TNBC compared to 50% in non-TNBC. In addition, hypermethylation of TWIST gene was insignificantly increased with increasing tumor size, <2 cm 33.3% and >2 cm 50% in TNBC compared to 54.2% and 32.3% in TNBC respectively (Table 3).
HIN-1 gene hypermethylation was significantly increased in TNBC by 65.7% in grade-III compared to 33.8% in non-TNBC. In TNBC, HIN-1 gene hypermethylation was increased significantly 74.3% in LN negative and insignificantly 57.8% in LN positive  compared to 36.8 % and 46.9%, respectively in Non-TNBC (Table 4). P16 hypermethylation was insignificantly increased in TNBC with tumor size from 27.8% in tumor size <2 cm to 46.2% in >2 cm and from 33.3% to 40.3% respectively in non-TNBC. In TNBC, P16 hypermethylation was associated with grade increasing 22.2% in grade-I compared to 47.2% in grade-II. With regard to LN status, in non-TNBC P16 hypermethylation was 36.8% in LN negative and 46.9% in LN positive while in TNBC no association was observed (Table 4).

Discussion
Although several studies have demonstrated that DNA hypermethylation plays an important role in breast carcinogenesis (Cho et al., 2009;Tan et al., 2012), little information is available about DNA methylation status in TNBC. Accordingly, this study investigates the differential methylation of the promoter region in specific group of genes in TNBC and Non-TNBC Saudi's females. Our results detected overall higher methylation frequencies of GSTP1, HIN-1, TWIST, ID4, PAX5 and DAPK genes in TNBC compared to non-TNBC. The promoter hypermethylation of these genes can be used as tumorspecific biomarker in TNBC Saudi's patients.
Death associated protein kinase (DAPK) gene is a positive mediator for programmed cell death induced by γ-interferon (Suijkerbuijk et al., 2010). The loss of DAPK gene expression was associated with aggressive and metastatic phenotype in many tumor types primarily by promoter hypermethylation (Suijkerbuijk et al., 2010). In the present study, DAPK gene was hypermethylated at highest frequencies in TNBC compared to non-TNBC. In addition, we found strong and higher association between DAPK hypermethylation and tumor grade and size in both TNBC and non-TNBC. Similarly in patients with head and neck cancer, Sanchez-Cespedes et al. (2000) observed an association between DAPK methylation and tumor size (Sanchez-Cespedes et al., 2000). They also found frequently lost in the expression of DAPK gene and protein in human cancer often as a result of silencing by DNA methylation. Similarly, higher hypermethylation of DAPK gene was observed in more advanced stage of cervical cancer (Narayan et al., 2003). Accordingly, one can anticipate that this hypermethylation of DAPK gene with the consequent lose of its expression may play a role in breast cancerinogenesis.
ID4 gene has regulative functions for cell differentiation and growth. Inactivation of ID4 gene via promoter hypermethylation has been shown in human tumors such as breast cancer (Noetzel et al., 2008). Data presented here demonstrated differential increase of ID4 hypermethylation in TNBC than Non-TNBC cases. In addition, the incidence of ID4 hypermethylation was increased with increasing tumor size and the number of LN positive in both TNBC and non-TNBC cases. Accordingly, our results suggested that hypermethylation of ID4 as a potential tumor suppressive gene may play an important role in BC development and can be used as a genetic marker for prediction of early metastasis that could explain the aggressiveness of TNBC compared to non-TNBC. Our results are consistent with the data presented by Umetani et al. which have demonstrated a significant lower mRNA level of ID4 in primary breast cancer specimens and indicated that the ID4 transcription was inactivated by promoter hypermethylation in clinical breast cancer specimens (Umetani et al., 2005).
Glutathione S-transferase P1 (GSTP1) is a polymorphic gene encoding active, functionally different variant proteins that are thought to function in xenobiotic metabolism and play a role in susceptibility to cancer, and other diseases. As different types of diet has been implicated in breast cancer, the loss of GSTP1 secondary to hypermethylation lead to impaired of cellular defenses leading to increasing genome damage and cancer  :http://dx.doi.org/10.7314/APJCP.2015.16.2.541 Increased DNA Hypermethylation in Triple-negative Breast Cancer development (Wang et al., 2014. In our study, high frequency of GSTP1 hypermethylation was significant associated with TNBC. This finding is important as GST gene is a family of enzymes that detoxify hydrophobic electrophiles, which include carcinogens that have been occupied with variety of cancers (Wang et al., 2014). The detection of GSTP1 hypermethylation was not correlated with other clinical parameters such as age, tumor size and LN status which are consistent with previous study (Jentzmik et al., 2012). Hypermethylation of GSTP1 with high frequency in different tumor grade was pathologically correlated with early stage of cancer.
In human breast cancer, hypermethylation of tumor suppressor gene is clearly observed. HIN-1 gene, a putative cytokine, is down-regulated in breast cancer tissues by promoter hypermethylation (Park et al., 2011). The present study showed increased in the frequency of HIN-1 gene hypermethylation in TNBC than Non-TNBC patients with higher incidence in LN positive in both groups. Other study found frequent methylation of HIN-1, CDH13 and RARβ2 genes in primary breast cancer and in metastatic LN but not in normal breast tissues (Feng et al., 2010). Our results showed higher hypermethylation of HIN-1 in both tumor grades II and III which indicates that HIN-1 gene is epigenetically at advanced stage in both TNBC and Non-TNBC and can be used as a good prognostic markers for Saudi's patients. Other found significant high frequency of HIN-1 gene promoter hypermethylation in tumor at an advanced stage compared to those at early stages (Lee et al., 2010).
PAX5 gene plays an important role in cell differentiation and embryonic development (Palmisano et al., 2003). In the current study, high non-significant frequency of PAX5 hypermethylation was observed in association with tumor grade, size and LN. In breast tumor, PAX5 gene hypermethylation is responsible for gene silencing. Moelans et al. found an association between PAX5 and tumor grade but no association with LN status (Moelans et al., 2011). In addition, PAX5 gene has been implicated in the pathogenesis of small lymphocytic lymphoma cancer and advanced-stage glioblastoma (O'Brien et al., 2011). DNA methylation was linked to the transcriptional silencing of PAX5 genes in murine myeloma cells (Danbara et al., 2002).
TWIST gene belongs to the basic-helix-loop-helix family of an antiapoptotic and prometastatic transcription factors (Sung et al., 2011). TWIST gene can act as oncogene that inhibits apoptosis in a p53-independent manner and may be important to the biology of tumor distant metastasis (Escobar-Cabrera Je, 2013). It may also alter cellular growth via its effects upon chromatin structure (Hamamori et al. 1999). TWIST hypermethylation was increased with tumor size increasing in TNBC and decreased in Non-TNBC. Other study suggested that there is no association between the tumor size and TWIST gene in invasive breast carcinoma patients (Bae et al., 2005).
P16 is a well-documented tumor suppressor gene in many tumors (Peurala et al., 2013). Promoter methylation of P16 has been reported in different studies (Demokan et al., 2012;Jha et al., 2012;Khor et al., 2013;Wani et al., 2013). In the present study, P16 hypermethylation showed insignificant increase in both TNBC and Non-TNBC and was associated with tumor grade increasing in TNBC. Similarly, Radpour et al. observed a correlation between P16 gene hypermethylation and stage of cancer (Radpour et al., 2011). We suggested that P16 gene may not only play a significant role in early stage of BC but also in cancer progression. This is clear in non-TNBC in which the P16 hypermethylation was associated with LN status and was insignificantly increased in tumor size. Other study by Zhao et al. found no correlation between size of cancer, LN metastasis and methyaltion rate of p16 gene .
Cyclin D2, a member of D-type cyclins, is implicated in cell cycle regulation, differentiation and malignant transformation (Sakuma et al., 2007). In the current study, Cyclin D2 gene promoter is hypermethylated higher in both BC groups than in benign breast tissue and it was significantly increased with tumor grade in non-TNBC. Previous findings on invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) showed a trend towards increased Cyclin D2 methylation progressing from lowgrade ductal in situ (DCIS) breast cancers grades-I and -II 27% to high-grade DCIS 39% to IDC 52% (Evron et al., 2001;Lehmann et al., 2002).
CDH13 gene can act as a tumor suppressor gene and its expression is decreased in invasive carcinomas resulting in decreasing cell-cell adhesion enhancing tumor progression and invasion (Ellmann et al., 2012). We reported a significant increase in CDH13 gene hypermethylation in TNBC compared to Non-TNBC and was increased in LN positive TNBC cases. This increase is due to the association between this gene with the hormone receptor (Feng et al., 2007). Similarly, more frequently CDH13 hypermethylation was observed in invasive adenoma than in non-invasive adenoma and was associated with high grade (Kornegoor et al., 2012). Also, in breast cancer cell line higher hypermethylation of CDH13 was found and rare in non-malignant and control tissues (Jung et al., 2013). Therefore, the suppression of CDH13 gene by methylation is associated with tumor formation and can be used as a marker for breast cancer development and invasion. RARβ1 is involved in regulation of cellular growth inhibition and apoptosis. Our study established that RARβ1 promoter is frequently hypermethylated in both TNBC and non-TNBC. RARβ1 gene mediates the growth inhibitory effects of retinoic acids in breast cancer cells and also several studies established RARβ1 gene promoter hypermethylation in breast carcinoma (Raffo et al., 2000;Weiwei et al., 2007). Hypermethylation of RARβ was correlated with HER2-positive tumors and with poor prognosis (Mehrotra et al., 2004). RARβ gene can act as a tumor suppressor gene and loss of its expression is found in variety of tumors (Liu et al., 2011). These data provide evidence that hypermethylation is the major mechanism involved in RARβ gene silencing which resulted in impaired RARβ function during BC development.
In conclusion, data from this study suggest that the epigenetic event clearly observed in TNBC and Non-TNBC patients might be used as marker for Saudi's BC. Moreover, this study highlights the promotor methylation of specific genes involved in different molecular pathways, detoxification, proapoptotic gene that potentially inhibits metastasis, tissue differentiation, regulate expression of tissue-specific genes, tumor suppressor gene and transcription factor. Information from this study may be useful in epigenetic therapy for breast cancer.