Correlation between the DNA methylation and gene expression of IGFBP5 in breast cancer

Abstract

BACKGROUND:

The insulin-like growth factor binding protein5 (IGFBP5) is often dysregulated in human cancers and considered neither a tumor suppressor nor an oncogene.

OBJECTIVE:

We aim to examine the reason of the changeable gene regulation of IGFBP5 in the case of methylation in breast cancer.

METHODS:

We used methyl-specific polymerase (MSP) chain reaction to detect CpG methylation of IGFBP5 promoter and exon-I in breast cancer and adjacent tissues. Gene expression is evaluated by quantative polymerase chain reaction (qPCR).

RESULTS:

IGFBP5 methylation was detected in 24 of 58 (41%) and 54 of 56 (96.5%) promoter and exon-I site respectively in tumor tissues. In adjacent tissues 17 of 58 (29%) and 53 of 56 (96.5%) was methylated. IGFBP5 expression was higher estrogene receptor (ER)(+) than ER(−) patients (p = 0.0549). Beside, we found a positive correlation between the expression of IGFBP5 and G2 tumor grade (p = 0.0131). However, no correlation was observed between IGFBP5 expression and age, menopause or the presence of lymph node metastasis (p > 0.05).

CONCLUSIONS:

In summary, our results showed that IGFBP5 promoter and exon-I methylation did not have any differences between tumor and adjacent tissues so that IGFBP5 methylation did not change IGFBP5 gene regulation in breast cancer. This is the first study investigating the IGFBP5 gene methylation in breast cancer.

Keywords

IGFBP5 breast cancer methylation MSP promoter

Introduction

Breast cancer is the most frequent carcinoma worldwide and the second most common cause of cancer-related mortality in women [1]. Genetic and epigenetic alterations of proto-oncogenes and tumor suppressor genes may define the faith of cells converted to cancerous [2,3]. Epigenetic alterations affecting the gene regulation direct DNA methylation, histone modifications, miRNA expression, and nucleosome positioning and higher order chromatin. The importance of epigenetic changes has been realized in many malignancies, including breast cancer [2]. Epigenetic disregulation of genes associated with cell cycle, apoptosis, DNA repair and cell adhesion leads to tumor formation, its progression, and drug resistance in breast cancer [4]. Lots of genes involved in invasion and metastasis, enabling replicative immortality, evading growth suppressors, sustaining proliferative signaling, inducing angiogenesis are silenced by DNA methylation [5]. DNA methylation, partially on the 5 position of the pyrimidine ring of cytosines in the context of CpG dinucleotides, is the most prevalent genetically programmed DNA modification [6] and aberrant promoter methylation is initiated at 1% of all CpG islands, and as much as 10% become methylated during the tumorigenesis [7]. Beside, CpG islands found in gene body, are more susceptible to aberrant methylation than the respective promoter sequences during cancer process [8]. However, methylation of these CpG islands does not necessarily diminish transcription. DNA methylation might be a useful biomarker for tumor diagnosis and/or risk assessment.

The IGF (insulin-like growth factor) axis plays a crucial role in the regulation of cellular growth and differentiation, developmental processes and malignant cell transformation [9,10]. IGF binding protein (IGFBPs) members of IGF axis modulate the bioavailability of the IGFs that are potent mitogenic and survival factors for both normal and cancer cells through binding IGFs and so controlling of IGF-I and IGF-II induced proliferation [11]. IGF system members, including the IGFs, IGF-IR and IGFBPs, have important roles in the development and progression of breast cancer. IGFBP-5, an abundant and a conserved IGFBP, is a secreted protein that can also translocate to the nucleus utilizing nuclear localization sequence [12]. In humans, IGFBP5 is 29 kDa protein and has 252 amino acids. IGFBP5 not only regulates vital function of cell; cell growth, differentiation, apoptosis, adherence, and movement but also has important role in tumor growth [13]. IGFBP5 is reported to inhibit the proliferative responses of breast cancer cells to IGF-I [14]. Wang et al. displayed a significant correlation between overexpression of IGFBP5 and the histologic grade of ovarian cancer and glioblastoma [15,16]. In activated stellate cells and myofibroblasts, IGFBP5 decreases apoptosis via an IGF1-independent mechanism and enhanced survi [17].

It is found that IGFBP5 expression level is associated with tumorigenesis/metastasis in several cancer types including breast [15]. In breast cancer, IGFBP5 has shown crucial upregulation in lymph node metastases relative to their paired primary cancers [18]. Bo Young Ahn [19] revealed that in breast cancer, low IGFBP5 expression was associated with shorter overall survival after tamoxifen therapy.

Although there are many studies mentioned functions of IGFBP5 in healthy and cancerous tissues, no study has investigated methylation of IGFBP5 gene in breast cancer. To date this is the first study that evaluate the methylation of IGFBP5 promoter and exon-I in breast cancer. In the present study, we used MSP to study the methylation status of IGFBP5 gene promoter and exon-I in breast cancer and adjacent tissues to evaluate its impact on gene expression and the clinical significance of IGFBP5 in the diagnosis and prognosis of breast cancer. To explore the methylation status of IGFBP5 gene promoter and exon-I 57 breast cancer tissues were used. Gene expression was evaluated via real time PCR.

Materials and methods

Patient samples and histopathologic evaluation

Samples of 57 breast cancer patients, who diagnosed at the Department of General Surgery of the School of Medicine, Marmara University from July 2010 to January 2012, were utilized in this study. Age of the patients differ from 35 to 76. The Ethics Committee of the Marmara University approved the study and informed consent from all subjects were taken. Tumor histology and tumor grade were evaluated at primary diagnosis and were extracted from pathology reports. Tumors were graded according to the Bloom- Richardson grading modified system [20]. Human epidermal growth factor receptor 2 (Her2) status was designed by immunohistochemical analysis using Dako HercepTest kit (Dako, Carpinteria, CA). For the Her2 score of 2, further fluorescence in situ hybridization was performed [21]. For progesterone receptor (PR) status, the PR monoclonal antibody PgR 636 (Dako, Wiesentheid, Germany) was used; for ER status, the ER monoclonal antibody clone SP1 (Neo Markers, Fremont, CA) was used.

Tissues and DNA preparation

Breast cancer and adjacent tissue samples were obtained from patients diagnosed with breast carcinoma. Clinical and pathological information, including age, ethnicity, menopausal status, type of tumor, disease stage, axillary lymph nodes, tumor size and biomarkers, were collected. Genomic DNA was isolated using a purification kit (AllPrep® DNA/RNA/Protein Mini, Qiagen, Darmstadt, Germany) from fresh tissues and were frozen at − 20^∘ C until use.

Bisulfite treatment and methylation specific PCR (MSP)

Methylation of the IGFBP5 promoter and exon-I was evaluated by bisulfite treatment. This treatment leads to a chemical conversion of any unmethylated cytosine to uracil, while the methylated cytosine remains unmodified. Four primer pairs were designed to differentiate methylated DNA from unmethylated DNA in the case of two regions.

Total genomic DNA from tumor tissues and adjacent tissues were modified by EpiTectPlus DNA Bisulfite Kit (Qiagen, Darmstadt, Germany). The bisulfite-treated DNA was used as the template for PCR, using fast start pcr master mix (roche, Germany). These methods were applied by the manufacturer’s instructions. For bisulfite conversion, approximately 1 μ g of DNA from tumor and adjacent tissues were treated with sodium bisulfite for 5 hours utilizing the EpiTectPlus DNA Bisulfite Kit (Qiagen, Darmstadt, Germany) and then DNA was cleaned with same kit.

To determine methylation patterns of CpG islands in IGFBP5 gene promoter and exon-I site, methylation-specific PCR analysis was successfully performed for 57 samples. Epigenetic regulation by methylation gene specific manner is suggested by the presence of a CpG island in the regulation site of genes [22]. The sequence of the human IGFBP5 gene was retrieved from the National Center for Biotechnology Information (NCBI) gene database. GC content of IGFBP5 promoter and exon-I region, that might be an indication of the presence of CpG islands, were analyzed. This GC rich region contains two CpG islands (NCBI Reference Sequence: NC_000002.12, >g i|568815596:c216695589-216695243 and (NCBI Reference Sequence: NC_000002.12, > g i|568815596:c216694609-216694439). The first CpG island is studied as it contains a transcription start site and transcription factors binding sites. In addition, first CpG island involves promoter and exon-I sequences of IGFBP5 [23] (Fig. 1).

MSP primers were designed using Methyl Primer Express® Software [24] (Table 1). All primers were purchased from IDT (Leuven, Belgium). Polymerase chain reaction amplification was performed using fast start pcr master mix (roche, Germany). 1 μ l DNA from bisulfite treated samples and control DNA as template were used for MSP. In final volume of reaction (50 μ l) PCR primer and DNA concentrations were 0,4 μ M and 10 ng/μ l, respectively. The PCR program used was 95^∘ C for 10 min, 35 cycles of 94^∘ C for 15 s, 55^∘ C for 30 s, 72^∘ C for 30 s and 72^∘ C for 10 min as a final extension. Products were visualized in a 2% agarose gel and compared with positive (universally methylated DNA; Qiagen) and negative controls [25].

Real time PCR

Expression level of randomly selected 25 patients IGFBP5 in breast cancer patients were detected using real time pcr method. Transcriptor High Fidelity cDNA synthesis kit (Roche, Germany) was used for cDNA synthesis with 500 ng of total RNA in a reaction volume of 20 μ L. Real time quantitative PCR for IGFBP5 and beta actin were performed using Light Cycler 480 Probes Master (Roche, Germany). 2.5 μ L of cDNA was used in a reaction volume of 10. All reactions were performed in duplicates for the reference housekeeping genes, beta actin and IGFBP5. Analysis and quantification was performed using Light Cycler 480 software. The level of IGFBP5 gene expression was normalized to that of the internal control (β -actin) and was determined by the 2^−ΔΔCT method.

Statistical analysis

Variables that were observed statistically significant by the univariate analysis were included in the final multivariate Cox regressive analysis. P-values of less than 0.05 were considered statistically significant for single testing, whereas statistical significance was set at lower threshold (p < 0.01 or p < 0.025) for multiple testing. Statistical data were obtained by using Statistical Package for the Social Sciences (SPSS) for Windows version 16.0 (SPSS Inc., Chicago, Illinois, USA). The mean 2^−ΔΔCt values between tumors and adjacent normal tissues were compared by both the paired and unpaired t test. The associations between methylation of IGFBP5 and age, ER/PR status, stage of disease, and histologic grades were determined by the Fisher exact test, the x² test, and the x² test for trends. All tests were 2-tailed, and results were considered significant when p value was < 0.05.

Results

IGFBP5 promoter and exon-I are differentially methylated in breast cancer patient samples

Methylated and unmethylated MSP products were acquired on the polyacrylamide gel. The presence of MSP band was considered as methylated regardless the band intensity in this study. Methylated (M) and unmethylated (U) band profiles for promoter and exon-I of IGFBP5 in tumor and adjacent tissues were displayed in Fig. 2. 24 (41%) tumor tissues and 17(29%) adjacent tissues of 58 cancer patients for exon-I site and 54 (96,5%) tumor tissues and 53 (94,5%) adjacent tissues of 56 cancer patients for promoter site has shown to be methylated.

Allelic methylation patterns were different between promoter and exon-I site. No biallelic methylation (M/M) signals were detected for exon-I site; but 7 samples for promoter site (Table 2). Methylation patterns were not meaningful statistically. No significant methylation pattern was observed in neither the normal nor breast cancer tissue samples.

IGFBP5 DNA methylation and gene expression

IGFBP5 expression levels of tumor tissues and adjacent tissues from breast cancer patients were evaluated. Randomly selected 25 breast cancer samples within our study group were involved in IGFBP5 expression analysis (Fig. 3). According to 2^−ΔΔCT values 11 of 25 (44%) tumor tissues showed higher expression than adjacent tissues, 14 of 25 (56%) adjacent tissues showed higher expression than their cancerous tissues. Three patients, having higher IGFBP5 expression in tumor tissues, also showed biallelic methylation in promoter site in tumor tissues. However, 2 of them also showed the same methylation pattern in adjacent tissues. No significant correlation between IGFBP5 expression level and methylation patterns of IGFBP5 promoter and exon-I site was observed (p > 0.05).

Expression of IGFBP5 mRNA in breast cancer was determined by qPCR. The median of IGFBP-5 expression was determined and the results higher than the median were considered highly expressed, the results below the median were considered lowly expressed.

IGFBP5 gene methylation pattern neither promoter nor exon-I site and IGFBP5 mRNA expression did not have any meaningful correlation (p > 0.05).

Relation between IGFBP5 methylation and clinicopathological characteristics in breast cancer

Several studies emphasized the correlation between presence of IGFBP5 protein and clinicopathological characteristics in breast cancer [26]. IGFPB5 expression was found higher in breast cancer than benign breast epithelium in reported studies. Methylation status influences the protein expression. In this study, IGFBP5 methylation status have not been altered according to clinicopathological factors. No significant correlation was found between IGFBP5 methylation status and breast cancer subtypes (Luminal A, Luminal B, HER2, Luminal-HER2 and basal type), tumor grade, hormone receptor status, ki67 expression, tumor stage or node status. However, a positive correlation was found between methylation of IGFBP5 exon-I and postmenopausal status (p = 0.0326).

The results of the MSP analysis of IGFBP5 promoter and exon-I methylation in the 57 breast cancer patients are summarized in Table 3.

Effect of IGFBP5 gene mRNA expression of clinicopathological characteristics in breast cancer

The correlation between IGFBP5 expression and the clinicopathological parameters, including age, disease stage, lymph node status, tumor grade, size and expression of ER, PR and Her-2/neu, were analyzed. The results revealed a positive correlation between ER status and IGFBP5 mRNA expression. ER (+) patients showed higher expression of IGFBP5 mRNA comparing to ER (−) patients (p = 0,0549). Patients divided into two groups, as one showed high IGFBP5 expression and the other showed low depending on median score. Evaluating IGFBP5 expression in high IGFBP5 group showed that best part of patients (46%) is in G2 stage. Beside that 38% of patients are in G3 stage and 8% in G1 stage. There was a positive correlation between the expression of IGFBP5 and G2 tumor stage (p = 0.0131). However, no correlation was observed between IGFBP5 expression and age, menopause or the presence of lymph node metastasis (p > 0.05).

Survival analysis

One of the followed patients has died. Tumor tissue mRNA of death patient was higher comparing the adjacent tissue. IGFBP5 promotor site was methylated but exon-I site was unmethylated for this patient.

Discussion

Breast cancer is the most frequent carcinoma in females, and the second most common cause of cancer related mortality in women [1]. IGFBP5 is a member of IGF axis restrict IGFs action which modulates breast cancer cells [11]. It has been repeatedly shown to regulate breast cancer behavior both in vivo and in vitro and correlated with a poor prognosis in cancer patients [14,26]. In this study, we revealed that IGFBP5 promoter and exon-I methylation and its effect on gene expression and clinicopathological factors in breast cancer.

IGFBP5 has been shown to affect cell proliferation and apoptosis differently. IGFBP5 has been shown to inhibit proliferation of breast cancer in vitro and in vivo [14]. Beside that IGFBP5 was found at higher level in intrahepatic cholangiocarcinoma than normal liver tissue [27]. Due to the nature of its changeable expression pattern and effects in both healthy and cancer tissues, it is considered neither a tumor suppressor nor an oncogene [28]. Li et al. showed that IGFBP5 mRNA overexpression may play a positive role in breast tumorigenesis and development [29]. In this study, we did not find any meaningful correlation between IGFBP5 mRNA expression and breast cancer progression.

Breast cancer is a hormone-dependent disease. ER/PR status reflects the level of estrogen and progesterone in vivo, and is a predictor of clinical outcome of breast cancer patients. Li et al. showed that IGFBP5 mRNA was down-regulated in ER/PR (−) patients compared to ER/PR (+) patients. Beside, they suggested that IGFBP5 upregulation could not be a prediction factor for the disease-free survival of node negative breast cancer patients [29]. A positive correlation was revealed between the expression of IGFBP5 protein ER/PR status in axillary lymph node-negative breast cancers [30]. In addition, we found a correlation between ER (+) patients and IGFBP5 mRNA expression (p = 0,0549). Patients with ER (+) status showed higher IGFBP5 mRNA expression than ER (−) patients. On the other hand, no significant differences of IGFBP5 were found between the mRNA level of IGFBP5 and tumor size, clinical stage, nuclear grade, PR status and Her2 status.

Epigenetic changes on genes are known as an early hallmark of carcinogenesis. In last decades many studies revealed the importance of promoter and exon methylation on gene expression in several cancers. The disregulation of genes leads to tumorigenesis initiation and progression. Genetic and epigenetic changes may result in upregulation and/or downregulation of genes implicated in cell proliferation [31]. DNA methylation in promoter may has an impact on the transcription directly by blocking the binding of transcriptional activators [32]. Although there is no data yet about IGFBP5 methylation, many studies were reported for the other IGF axis members. Two closely related members of the IGFBP superfamily, IGFBP-rP1 and IGFBPL1, were found to be subjects to frequent methylation-dependent transcriptional silencing in ductal breast cancers. Both genes were unmethylated and expressed in normal breast epithelium. Furthermore, they reported that aberrant cytosine methylation in the transcriptional regulatory elements of both genes caused down-regulation and/or absence of expression of IGFBP-rP1 and IGFBPL1 in breast cancer cell lines and primary ductal carcinomas [33]. Aberrant DNA methylation and histone modifications make silence many tumor suppressor genes in human cancers [31,34]. Dar et al. reported that expression of IGFBP3 mRNA in human primary melanomas was low when compared to healthy samples. When applying 5-AZA to melanoma cells that caused increasing in acetyl H3, acetyl H4, 2H3K4 and 3H3K4 histones compared with untreated control in melanoma cell lines. Enrichment of these active chromatin modifications near the transcription start site of the IGFBP3 gene is associated with active gene expression [34]. The other member of IGF axis, IGFBP7, found methylated in the prostate cancer cell lines, prostate cancer and high grade prostatic intraepithelial neoplasia compared to benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia samples and they revealed aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in CaP (prostate cancer) cell lines [31]. In addition a study of colon cancer showed that methylation dependent regulation of IGFBP7 expression occurred in the first exon and intron, and was independent of the methylation status of the 5^′ end of the promoter [35]. Another IGFBP7 methylation report was revealed for oesophageal adenocarcinoma and Barrett’s oesophagus cell lines. IGFBP7 methylation, was correlated with IGFBP7 decreasing transcriptional expression, was observed higher in oesophageal adenocarcinoma and Barrett’s oesophagus cell lines than in adjacent histologically normal squamous mucosa [36]. In this study we did not found any correlation with IGFBP5 promoter and exon-I methylation between tumor and adjacent tissues. Methylation pattern did not change the IGFBP5 gene expression in our study.

Aberrant methylation of CpG sites in promoter and outside the promoter regions may also influence the binding of regulatory molecules and thus gene expression. It is also possible that methylation of IGFBP5 promoter and exon-I site alone may be insufficient to show IGFBP5 methylation pattern in breast cancer. IGFBP5 has two CpG islands [23]. In our study, we explored promoter and exon-I site of IGFBP5 gene methylation. However for some genes intergenic CpGs may be more effective than CpG in promoter during the gene regulation. More studies will help to make gene regulation of IGFBBP5 more clear that is impressed by DNA methylation in breast cancer. Given the integral role of histone deacetylation and chromatin remodeling in gene silencing would be interesting to determine a complete epigenetic profile of IGFBP5 gene and determine their contribution to the transcriptional silencing of this important tumor suppressor gene in breast cancer.

IGFBP5 has a major role development and progression in breast cancer. Detection of abnormal changes of IGFBP5 methylation pattern in early stage of breast cancer patients could be useful for early detection of breast cancer. IGFBP5 methylation studies are carried out with increased number of populations and using breast cancer cell lines and/or healthy people samples it will provide a stronger rationale evidence for development of new therapeutic and diagnostic approaches for breast cancer patients. Early detection of cancer development is crucial for care it and methylation changes of several genes happen in precancerous tissues. So revealing methylation pattern of genes will save patients and shrink economical deprivation paid for medicine and medical therapy.

In summary, this is the first report indicating the DNA methylation pattern of IGFBP5 promoter and exon-I in breast cancer. We found that methylation pattern of IGFBP5 promoter and exon-I was not effective on the gene expression. IGFBP5 gene sequence has two CpG islands and we studied first island, therefore we can speculate that second CpG islands methylation pattern may be more effective than the first CpG island on IGFBP5 gene expression. Further studies need to clarify the importance of the second CpG island on the gene expression. Beside that, IGFBP5 exon-I methylation and postmenopausal status of patients were found to be relevant in this study. Our present data indicated that IGFBP5 gene expression correlated with ER status and tumor grade in breast cancer. However additional research is needed to clear up epigenetic changes of IGFBP5 regulating gene expression that is involved in the pathogenesis of breast cancer.

Footnotes

Acknowledgements

This work was supported by grant (SAG-C-DRP-060911-0275) from Marmara University, Scientific Research Research Projects Committee and (SBAG-111S161 to MA) from the Scientific and Technological Research Council of Turkey (TUBITAK).

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