Cancer Letters

Cancer Letters

Volume 237, Issue 2, 18 June 2006, Pages 272-280
Cancer Letters

DNA hypermethylation in breast cancer and its association with clinicopathological features

https://doi.org/10.1016/j.canlet.2005.06.011Get rights and content

Abstract

Aberrant hypermethylation of gene promoter regions is one of the mechanisms for inactivation of tumour suppressor genes in breast cancer. We investigated whether hypermethylation identifies breast cancers with distinctive clinical and pathological features. We evaluated the methylation of RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST in 193 breast carcinomas. Methylation frequencies ranged from 11% for CCND2 to 84% for ER. Tumours with frequent methylation (4–6 genes) were more often poorly differentiated compared to those with infrequent methylation (0–2 genes; P=0.004). Tumours with ER and CDH1 methylation were associated with significantly lower hormone receptor levels, younger age at diagnosis and the presence of mutant p53. Our data suggests that gene methylation may be linked to various pathological features of breast cancer, however, this requires confirmation in larger studies.

Introduction

Breast cancer is the single most common cause of cancer-related mortality in women worldwide, claiming almost 400,000 lives per year [1]. Similar to other tumour types, breast carcinoma is thought to arise following the activation of oncogenes and inactivation of tumour suppressor genes. It is becoming increasingly recognized that methylation-induced transcriptional silencing of tumour suppressor genes is a major epigenetic mechanism leading to the inactivation of these genes. Methylation of CpG dinucleotide-rich areas in gene promoter regions is thought to be especially relevant for the silencing of important growth control genes [2]. For breast cancer, some of the genes reported to undergo hypermethylation are involved in cell cycle regulation (p16, CCND2), cell adhesion (CDH1), DNA repair (BRCA1) and cell signalling (ER and RARβ2) [3], [4]. Identification of methylated gene targets has helped to elucidate the molecular pathogenesis of breast cancer by indicating which growth control pathways might be affected. Studies on tumour-specific DNA hypermethylation may also have clinical value for the early detection of breast cancer using ductal lavage fluids.

One of the major issues in the study of DNA hypermethylation patterns is whether they identify tumour subgroups having distinctive biological properties that could be used for prognostication and for prediction of response to therapy. For colorectal cancer, the concurrent methylation of promoter regions in certain genes including p16, MLH1 and MDR1 is strongly associated with a tumour subgroup having characteristic clinicopathological features [5], [6], [7]. However, some authors have argued the microsatellite instability phenotype which arises following methylation-induced silencing of the MLH1 DNA mismatch repair gene is responsible for these features, rather than the DNA hypermethylation per se [8]. In breast cancer, microsatellite instability as defined by mutations of repeat sequences within coding regions is not observed [9], [10], [11] and hence the same argument cannot be applied to this cancer type.

There is some evidence in breast cancer that gene methylation might identify phenotypes with different histology or clinical properties. For example, a recent study using an array-based method found that poorly differentiated tumours exhibit more hypermethylated CpG islands than their moderate- or well-differentiated counterparts [12]. PTEN pseudogene methylation was associated with ERBB2 overexpression, larger tumour size and a higher histological grade [13]. Methylation of TWIST was reported to be more frequent in invasive ductal compared to invasive lobular carcinoma [14], while methylation of CCND2 varied according to tumour progression [15]. Others have reported significant associations between specific gene methylation and the response of breast cancers to hormonal and non-hormonal therapies [16]. However, in a studying involving 109 cases and 12 different genes, the authors concluded there was little evidence to support the existence of a distinct CpG island methylator phenotype in breast cancer [17].

The aim of the present study was to investigate for associations between gene hypermethylation and the major clinicopathological features of breast cancer, including prognosis. We studied the methylation of seven genes (RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST) in a large (n=193) and well characterized [18] series of primary breast cancer. These were chosen because of putative roles in breast cancer development or progression and because previous studies have shown they are commonly methylated in this tumour type (but not in normal breast epithelium) leading to down-regulation of expression [14], [15], [16], [17], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. The methylation-specific PCR (MSP) assay [39], reported to have a sensitivity of 1/1000 for the detection of methylated alleles, was used to identify tumour samples with hypermethylation in the promoter regions of the genes above.

Section snippets

Tumour samples

A total of 193 tumour samples from women undergoing surgery for primary invasive breast carcinoma at the Sir Charles Gairdner and Royal Perth Hospital in Western Australia between 1990 and 1994 were used in this study. The clinical and pathological features of this cohort have been described previously [18]. Genomic DNA was extracted from fresh frozen specimens using standard phenol–chloroform extraction procedures. Patient age at diagnosis ranged from 18 to 93 years (mean 60) and follow-up

Results

Representative examples of MSP results for each gene are shown in Fig. 1. Of the 186 tumour samples with results for all seven genes, 1 (0.5%) showed no methylation at any site, 16 (9%) were methylated at only 1 site, 39 (21%) at 2 sites, 61 (33%) at 3 sites, 43 (23%) at 4 sites, 16 (9%) at 5 sites and 10 (5%) at 6 sites. No tumour was methylated at all seven sites. Methylation frequencies ranged from 11% for CCND2 to 84% for ER (Table 2). For RARβ2, CCND2, p16 and TWIST the frequencies

Discussion

The aim of this study was to determine whether the methylation of genes with a putative role in breast cancer were associated with distinctive pathological characteristics including prognosis. The idea of a distinctive phenotype associated with aberrant DNA hypermethylation (CIMP+) was first put forward for colorectal cancer [5]. However, some workers have argued that the characteristic features of CIMP+ colorectal cancers are due to the MSI+ phenotype that arises following methylation-induced

Acknowledgements

This work was supported by a grant from the Cancer Council of Western Australia. The authors are grateful to Fabienne Grieu for technical advice.

References (44)

  • J.G. Herman et al.

    Gene silencing in cancer in association with promoter hypermethylation

    N. Engl. J. Med.

    (2003)
  • X. Yang et al.

    DNA methylation in breast cancer

    Endocr. Relat. Cancer

    (2001)
  • M. Toyota et al.

    CpG island methylator phenotype in colorectal cancer

    Proc. Natl Acad. Sci. USA

    (1999)
  • M. van Rijnsoever et al.

    Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands

    Gut

    (2002)
  • R. Anbazhagan et al.

    Microsatellite instability is uncommon in breast cancer

    Clin. Cancer Res.

    (1999)
  • E. Forgacs et al.

    Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers

    Oncogene

    (2001)
  • C. Adem et al.

    Microsatellite instability in hereditary and sporadic breast cancers

    Int. J. Cancer

    (2003)
  • P.S. Yan et al.

    CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer

    Clin. Cancer Res.

    (2000)
  • J.M. Garcia et al.

    Promoter methylation of the PTEN gene is a common molecular change in breast cancer

    Genes Chromosomes Cancer

    (2004)
  • M.J. Fackler et al.

    DNA methylation of RASSF1A, HIN-1, RAR-beta, Cyclin D2 and Twist in in situ and invasive lobular breast carcinoma

    Int. J. Cancer

    (2003)
  • M. Widschwendter et al.

    Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen

    Cancer Res.

    (2004)
  • Y.K. Bae et al.

    Hypermethylation in histologically distinct classes of breast cancer

    Clin. Cancer Res.

    (2004)
  • Cited by (91)

    • Modulation of epigenetic methylation enzymes by synthetic and natural agents

      2023, Transcription and Translation in Health and Disease
    • Cell free DNA biology and its involvement in breast carcinogenesis

      2020, Advances in Clinical Chemistry
      Citation Excerpt :

      Among the main ones, the following can be mentioned: advanced age, which allows the accumulation of somatic mutations, leading to telomerase dysfunction allowing gene silencing of several promoter regions; and exposures to increased estrogen concentrations that favor mammary epithelial cell growth, consequently leading to epigenetic DNA modifications [197]. Genes that undergo hypermethylation in breast tissue include growth genes involved in invasion and apoptosis (DAPK, TWIST1, HOXA5, RARβ2), cell cycle regulation (CDKN2A (p14ARF/p16INK4a), CCND2, RASSF1A), cell invasion and metastasis (CDH1 and APC), DNA repair (BRCA1 and GSTP1), and cellular signaling (ER and RARb2), among others [197–200]. Both global hypomethylation and promoter hypermethylation may accompany tumor development, and both have been recognized as common events in many cancers [194].

    • Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

      2014, Gene
      Citation Excerpt :

      In the same line with our results previous studies (Zhao et al., 2009; Wei et al., 2008; Parrella et al., 2004; Iwase et al., 1999) showed no correlation between ERα methylation and age. On the contrary, a study on Australian patients revealed that ERα methylation was associated with younger patients (Li et al., 2006). In our study, we did not see any correlation between ER3, ER 4 and ER5 methylation and HER2 receptor negativity, cancer type and cancer stage, These findings were in concordance with previous studies (Ramezani et al., 2012; Zhao et al., 2009; Izadi et al., 2012).

    View all citing articles on Scopus
    View full text