Hypermethylation is an epigenetic change that blocks the promoter region of a gene and results in gene silencing  . In breast cancer, tumor-related genes may be silenced by hypermethylation; many hypermethylated genes have been reported, and silencing of these genes plays important roles in carcinogenesis and tumor progression     . Identification of epigenetic changes and their correlation with other clinical factors could lead to improvements in cancer diagnosis and treatment.
Human basic helix-loop-helix DNA binding protein (TWIST; location: 7p21.2; GenBank: U80998) induces E-cadherin mediated cell-cell adhesion and induction of cell motility. Increased expression of TWIST correlates with tumor invasion and metastasis  .
DNA methylation is the epigenetic change, which has been mostly reported to be associated with TWIST and interfere with Twist expression. Usually, hypermethylation of the promoter region of TWIST gene has been known in certain cancers of breast, uterine cervix, ovary, bladder, colorectal, gastric, lung, bone and brain  . For the detection of cancer cells and cancer typing in biological samples, the tumor specific promoter hypermethylation of TWIST is becoming a promising tool. However, it is not very clear how the TWIST promoter hypermethylation is responsible for carcinogenesis. Evidences showed that the increased frequency of hypermethylated genes in distant metastasis might be an important event in cancer progression. It has been reported that the hypermethylated TWIST gene is more frequently found in the local and distant metastasis than in primary breast carcinomas  . To clarify the epigenetic mechanism of Twist, Gort  proposed two possibilities:  the proximal part of the TWIST promoter is not related to TWIST expression, rather than interfering with other genes in genomic proximity like HDAC9 and FERD3L;  hypermethylation of TWIST promoter might be an early event that precedes compensatory TWIST over-expression  . Chromatin remodeling is another important epigenetic change of Twist responsible for cancer. Evidence showed that a protein complex formed by Twist with the Mi2/nucleosome remodeling and deacetylase, termed as Twist/Mi2/NuRD, plays an essential role in invasive and metastatic cancer cells  . miR-10b has also been reported to target Twist but change of miR-10b expression alone can’t induce breast cancer cell into EMT  , and other factors might be associated. However, these mechanisms are not enough to understand the epigenetics of Twist in cancer clearly, and a vast things to know. In cancer therapeutics, controlling metastasis remains one of the mainstream focuses, and targeting Twist might be the new dimension in controlling metastatic cancers. Several attempts have been taken to manipulate Twist function in cancer cells, including epigenetic approaches. Particularly, in hormone resistant breast cancer, epigenetic modification of Twist is able to restore hormone sensitivity, as Twist can serve as a potential target for converting estrogen receptor (ER)-α-negative breast cancers to ER-α-positive breast cancers   . Exploring Twist epigenetics might be a very interesting topic for the cancer researchers for the invention of new therapeutic approach for cancer treatment.
The aim of this study is to find out the frequency and the level of methylation of TWIST gene in breast cancer to determine whether the methylated gene is associated with histopathological parameters. We have assessed whether DNA methylation profiling may be used clinically to distinguish the early diagnosis of cancer, tumor classification, prognosis and the regulation of treatment protocols.
2. Materials and Methods
2.1. Sample Collection and DNA Preparation
This study was conducted between 2009-2011. Formalin-fixed, paraffin-embedded samples from 80 primary breast tumors tissue samples were collected from Eskisehir Osmangazi University medical faculty of Oncology Clinic were included. Samples which were embedded in parafin tissue blocks as 10 sections of 10 microns thick and in eppendorf tubes were taken from Eskisehir Osmangazi University of medical faculty of Pathology department. Pathologists decided the sample size.All tumor specimens have at least 70% of tumorous tissue. Besides the TWIST methylation, the histopathlogical parameters such as age, tumor stage, tumor type, lymph node metastasis, estrogen receptor, progesterone receptor and Her2/neu status were collected from and related with the methylation levels. After deparafinization, genomic DNA was extracted using MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche) according to the manufacturer’s instructions.
2.2. Bisulphite Modification and Methylation Analysis
Approximately 250 - 500 nanogram of genomic DNA was bisulphite modified using the EpiTect Bisulphite kit (Qiagen) according to the manufacturer’s instructions. The modified DNA was eluted three times in 50 µl Elution Buffer (EB). Methylation analysis was performed using Methylation-sensitive high resolution melting (MS-HRM) in which the approximate level of methylation and presence of heterogeneous methylation can be determined in bisulphite modified DNA    . PCR amplification and high resolution melting analysis were performed on the Roche LightCycler 480 (Roche Applied Science, Laval, PQ, Canada) equipped with the Gene Scanning software (Version 1.5.0). MS-HRM primers and amplicon details are listed in Table 1. In order to detect the promoter methylation status of TWIST gene we used a real-time polymerase chain reaction (PCR) approach followed by high resolution melting curve analysis (HRM), considered as a rapid, highly sensitive and efficient method displaying the sequence-dependent melting profile of an amplicon. PCR and HRM analysis were consecutively performed on a LightCycler 480 (Roche Diagnostics GmbH, Germany) in one single run, and all samples were analysed in duplicate. Each reaction mixture contained ~1 - 2 µl of bisulfite-treated DNA, 200 nmol/µl of each primer, 10 µl of LightCycler 480 High Resolution Melting Master (Roche), 3.5 mM MgCl2 and PCR-grade water adjusted to a total volume of 20 µl. The conditions used in the Light Cycler 480 were an initial cycle at 95˚C for 10 min, followed by 50 cycles of 95˚C for 1 min, followed by annealing at 40˚C for 1 min. Samples were quickly warmed to 65˚C and then slowly warmed to 95˚C at 0.1˚C per second. HRM was performed from 65˚C to 95˚C with a temperature increase at 0.10˚C/sec  . CpGenome TM Universal Methylated DNA (Chemicon/Millipore, Billerica, MA) and CpGenome Universal Unmethylated DNA were used as the fully methylated and unmethylated controls, respectively. Methylation standards (25%, 50%, 75% and 100%) made by diluting the fully methylated control in the unmethylated DNA were used as controls. Whole-genome amplification (WGA) was also used to make a fully unmethylated control and performed as described previously  .
2.3. Statistical Analysis
The v2 test and binary logistic regression were used to analyse the relationship of methylation with the clinicopathological findings. The analyses were carried out with SPSS version 15.0 (SPSS Inc., Chicago, IL). A two tailed P-value of less than 0.05 was considered to be statistically significant for each comparison.
3.1. Patient Characteristics
Patient characteristics included in this study are summarised in Table 2. Patients with primary breast tumors were aged between 32 and 83 (median = 58.44 ± 11.09). Respectively 19/80 cases of them (24%) was ≤50 years of age, 61/80’s in (76%) > 50 years of age.
3.2. Methylation Status in Primary Breast Tumor Samples
A summary of the promoter methylation analysis of TWIST gene in 80 primary breast tumours are shown in Table 3 and Table 4 and examples of the MS-HRM results are shown in Figures 1-3. Mentioned gene showed detectable promoter methylation in primary breast tumours. TWIST1, was methylated in 25% of primary breast tumors.
3.3. Correlation between DNA Methylation and Clinicopathological Parameters
When the only methylated groups were compared, there is no correlation between the TWIST gene hypermethylation and tumor stage (p > 0.05) and age (p > 0.05). TWIST gene hypermethylation was found to be significant with lymph node positivity (p < 0.001), EstrogenReceptor (ER) positivity (p < 0.05), Progesterone Receptor (PR) negativity (p < 0.05), and HER2/NEU negativity (p < 0.05).
When we compare the hypermetylation of TWIST gene with the type of tumor, increased promoter methylation level have seen in invasive ductal carcinoma (IDC) then the invasive lobular carcinoma (ILC) tumor type (p > 0.001).
Table 1. Primer sequence of the gene.
Table 2. Clinical characteristics ofpatients.
Table 3. Associations between gene promoter methylation and clinicopathological features of breast cancer.
M: Methylation, UM: Unmethylation, n: Number of Patients.
Table 4. Methylation rates of TWIST gene and the prognostic factors of all cases.
UN: Unmethylated, ER: Estrogen Receptor, PR: Progesteron Receptor.
Figure 1. >25% of methylated sample seen with the control group in normalized and temperature shifted difference plot by gene scanning.
Figure 2. 75% of methylated sample seen with the control group in normalized and temperature shifted difference plot by gene scanning.
Figure 3. 100% of methylated sample seen with the control group in normalized and temperature shifted difference plot by gene scanning.
Aberrant promoter DNA methylation has been examined with various methodologies, including COBRA, SSCP, MSP and sequencing  . MSP is currently the most widely used method due to its claimed efficiency in heterogeneous cancer cell populations  . On the other hand it has limitations since it is time-consuming, more expensive and not quantitative in comparison with MS-HRM  . MS-HRM is a novel approach to identify aberrant methylation of gene promoter regions using sequence-dependent melting profiles of each amplicon  . It has been considered as the most rapid and sensitive in-tube method capable of detecting even 0.1% - 1% of DNA methylation in an unmethylated background, minimizing possible sample contamination, and requiring only low amounts of DNA template   . Furthermore, MS-HRM is a semi-quantitative method that has been claimed to distinguish homogeneous from heterogeneous methylation  .
TWIST belongs to the basic-helix-loop-helix family of transcription factors and is implicated in lineage-specific cellular differentiation and survival  . TWIST function in vertebrates governs early mesodermal patterning and osteogenesis  . Individuals with germ-line haploinsufficiency of the TWIST gene suffer from the hereditary disorder Saethre-Chotzen syndrome (acrocephakosyndactyly type III) characterized by premature craniosynostosis and limb, head, and face anomalies  . In cancer development, TWIST functions as a prometastatic oncogene. Expression of TWIST protein counteracts the proapoptotic effects of N-MYC by repression of p19ARF and thereby hampers TP53 function  .
At the DNA level, cancer development is characterized by genetic and epigenetic events. Methylation of CpG islands in promoter regions of tumor suppressor genes is a common epigenetic event  . Methylation abrogates proper TATA-binding protein binding to the promoter, leading to reduced expression of genes that play important roles in the cell cycle, cell adherence, cell signaling, DNA repair, and apoptosis  . Human breast carcinomas exhibit TWIST promoter hypermethylation at high frequency, ranging from 16% to 77%    . Moreover, methylation of the TWISTpromoter is a good predictor of human breast cancer presence  . These findings postulate methylation of the TWISTpromoter as an interesting breast cancer biomarker, but its functional significance remains unknown.
TWIST is a transcription factor that belongs to the basic-helix-loop-helix family and plays important role in cell differentiation and survival  . Hundreds of hypermethylated genes have been described in breast cancer. Scanlan et al, found 38% (37 out of 99) of Twist hypermethylation in breast cancer patients using quantitative multiplex methylation specific PCR (QM-MSP)  . In an another study conducted with Twist hypermethylation and type of tumor, they found Twist gene promoter was hypermethylated at a much lower frequency of 16% (3 of 19) in ILC compared to 56% (15 of 27) in IDC (p = 0.01)  . Gort et al. showed that promoter methylation of TWIST is significantly more prevalent in malignant compared to healthy breast tissue (34%)  . Shinozaki et al. showed promoter region CpG hypermethylation was identified in 151 primary breast tumors, TWIST promoter hypermethylation occurs (48%)  . Sunami et al. showed TWIST metyhlation (37.5%)  . Li et al. showed TWIST hypermethylation (59%)  . We found decreased promoter methylation level (25%). We though that this can be caused by the tumor heterogeneity.
Gort and Shinozaki et al. showed no correlation between clinicopathological parameters with TWIST gene promoter hypermethylation   . But Sunami and Li et al. showed methylation status of TWIST gene significantly higher in the ER-positive group   . Our study are consistent with the relationship between TWIST gene promoter hypermethylation and ER(+) with the Sunami and Li colleagues.
Our results showes increased level than the other researchers caused by the different technique. MSP is non-quantitative and even low amounts of background methylation will not be scored whereas MS-HRM can be used for semi-quantitative estimation of methylation and it’s more sensitive and spesifitive than the MSP.
Precision measurement, increasing every 0.1 to 1.0 degrees (˚C) caused by the temperature change of fluorescence signal intensity can be factored record refers to. Data retrieval, for every two seconds 0.1˚C to 1.0˚C increase in temperature of the fluorescent signal corresponds to is obtained by measuring the change in intensity. This is an indication of DNA denaturation has been a very good track. For this reason, the method is very high specificity and sensitivity. Very low rates of methylation differences can be detected.
In this study, we have demonstrated a significant association between methylated genes and known histopathological features in breast cancer patients in Turkey. We have shown that DNA methylation can be assessed even small quantities of FFPE DNA. Also MS-HRM is a powerful technique in molecular biology for the detection of epigenetic changes and can be used for semi-quantitative estimation of methylation. MS-HRM also is practically advantageous for use in diagnostics, due to its capacity to be adapted to high throughput screening testing.
Determining the epigenetic changes observed in cancer cells and increasing data related to the issue is gradually becoming important in terms of prevention of cancer, determination of prognosis and development of therapeutic approaches. This situation highlights the importance of methylation as a tumor marker.
Further examination of genes displaying high methylation levels in larger cohorts could provide for the early detection and/or disease monitoring and possibly for influencing therapeutic decisions.
O. E. identified cases; prepared samples; performed methylation assays; interpreted data and analyzed data; and wrote the manuscript. O. C. optimazed and performed methylation assays; conceptualized project; provided analysis; wrote and edited the manuscript. S. A. provided project oversight and coordination and analysis; wrote and edited the manuscript. B. D. A. identified cases; interpreted and analyzed data; wrote and edited the manuscript. All authors read and approved the final manuscript.
This work was financially supported by Eskisehir Osmangazi University Research projects (grant number: 201011037) and the study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Eskisehir Osmangazi University (number: 2010/173). The authors wish to thank Prof. Dr. Serap Isiksoy, Assistant Prof. Dr. Evrim Ciftci and the staff for prepairing formalin-fixed parrafin embeded tissues at Eskisehir Osmangazi University medical faculty of pathology department.
ER: Estrogen receptor; F: Forward; FFPE: Formalin-fixed, paraffin-embedded; HER2/neu: Human epidermal growth factor receptor 2; M: Methylation; UM: Unmethylation; MS-HRM: Methylation-sensitive high resolution melting;PCR: Polymerase chain reaction PR: Progesterone receptor; R: Reverse; TWIST: Human basic helix-loop-helix DNA binding protein; WGA: Whole genome amplified.