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. Author manuscript; available in PMC: 2020 May 18.
Published in final edited form as: Epigenetics. 2008 Mar 12;3(2):59–63. doi: 10.4161/epi.3.2.5899

Epigenetic gene silencing in the Wnt pathway in breast cancer

George J Klarmann 1,2, Amy Decker 1,4,†, William L Farrar 1,3,*
PMCID: PMC7233793  NIHMSID: NIHMS1574972  PMID: 18398311

Abstract

Breast cancer is one of the most common malignancies in women. Despite advances in treatment of endocrine-dependent tumors, the complete molecular basis of transformation is still unknown. What is clear is that a variety of genetic lesions and epigenetic modifications are present in the neoplasm. Disregulation of several signaling pathways is known to be associated with breast cancer development, among them is the wingless and integration site growth factor (Wnt) pathway. While genetic mutations of certain components of this pathway, such as APC, are significant contributing factors for colorectal cancers, they are typically not the predominate mechanism associated with breast cancer. Instead, it appears that DNA hypermethylation leads to aberrant regulation of the Wnt pathway in breast cancer, and as such, this review focuses on the epigenetic regulation of Wnt pathway components in breast cancer.

Keywords: breast cancer, DNA methylation, epigenetics, Wnt signaling, β-catenin, cancer stem cell

Introduction

Breast cancer is the most common cancer among women and is second only to lung cancer in mortality. According to the World Health Organization, approximately 1.2 million people worldwide were diagnosed with breast cancer in 2007. The American Cancer Society estimates for women that 213,000 new cases and 41,000 deaths will be attributed to invasive breast cancer in the United States this year. The chance of developing invasive breast cancer during a woman’s lifetime is approximately 1 in 8. Although much less common, approximately 1,700 cases of male breast cancer occur each year.

A combination of genetic and epigenetic changes, which lead to disregulation of genes and pathways controlling cell growth, contribute to breast cancer development. The two most common epigenetic alterations involving DNA methylation and histone modification result in changes in gene expression patterns without mutations in the gene. Though reversible, these modifications are maintained after DNA synthesis, resulting in heritable structural changes (reviewed by Widschwendter and Jones1). At the molecular level, DNA methylation is the enzymatic addition of methyl groups to the 5-position of cytosines that are most often located within CpG islands found in the promoter region of genes. Promoter hypermethylation effectively represses the transcription and subsequent gene expression, and occurs in many genes involved in breast cancer.1 Methylation is mediated by three known DNA cytosine methyltransferases, DNMT1, 3α and 3β.1 DNMT1 is the most abundant and is responsible for maintenance of established methylation patterns from the parent strand to the daughter strand in DNA replication. DNMT 3α and 3β are involved in de novo methylation and are up regulated in aging cells.1,2 DNMT1 and DNMT3β show increased expression in breast cancer.3,4

The histone modifications of acetylation, methylation, phosphorylation, polyADP-ribosylation and ubiquitination direct chromatin into transcription repressive or permissive configurations.2 For example, histone acetylation [through histone acetyl transferase (HAT)] allows protein access to DNA and favors transcription, whereas deacetylation by histone deacetylase (HDAC) enzymes, compacts the DNA into a transcription-repressive chromatin complex.

This review focuses on the Wnt signaling pathway and it’s epigenetic regulation in breast cancer. Unlike colorectal cancers,5 evidence for genetic mutations of Wnt pathway components such as adenomatous polyposis coli (APC) being an early and necessary step in breast malignancies is rare.6–9 However, various lines of evidence suggest that this pathway is disregulated in breast cancer,10–12 presumably through epigenetic mechanisms.

Wnt Signaling Pathway

The Wnt signaling pathway is important in cell differentiation and proliferation, cell movement and polarity, and for maintenance of self-renewal in hematopoietic stem cells (reviewed in ref. 13), and defects in this pathway are implicated in the pathogenesis of several tumor types, including breast cancer (reviewed in refs. 14 and 15). Thus, evidence is mounting that the Wnt pathway is a significant contributor to tumorigenesis. Wnt signals are transduced through two different pathways, the canonical and the non-canonical,13 largely due to the fact that Wnt is a family of closely related, secreted glycoproteins with at least 19 members that can bind to different cell surface receptors or different combinations of receptors that ultimately determine which specific pathway is activated.16,17 This review will focus primarily on the canonical pathway in which secreted Wnt signals are transduced through the Frizzled (FRZ) family of transmembrane receptors and the low-density lipoprotein receptor-related protein (LRP5/6) correceptor to ultimately stimulate β-catenin, the critical transcription regulator of growth-promoting genes (Fig. 1). There are numerous positive and negative regulators of this pathway, and loss of negative regulation, which activates constitutive signaling and growth, is an important contributor to tumor development.12,18–20

Figure 1.

Figure 1.

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Simplified cartoon of the Wnt signaling pathway. (A) In the absence of Wnt or if Wnt cannot bind Frizzled (FRZ) receptor due to sequestration by Wnt inhibitory factor (WIF-1) or frizzled-related protein (SFRP), β-catenin is phosphorylated and degraded, and control over differentiation and proliferated is maintained by the cell. (B) Wnt binding to FRZ and low-density lipoprotein receptor-related protein (LRP) clusters assembled on a Dishevelled (Dvl) scaffold allows β-catenin to accumulate in the cytoplasm and then translocate to the nucleus where, in complex with transcription factors TCF/LEF-1, various genes for proliferation and epithelial-to-mesenchymal transition (EMT) are activated.

Wnt binding to Frizzled is followed by Dishevelled (Dvl) aggregates assembling a scaffold-like structure at the plasma membrane.21 This triggers formation of clusters of LRP5/6 that are then phosphorylated by casein kinase I (CKIγ). Subsequently Axin, in complex with glycogen synthase kinase 3β (GSK-3β) and the associated APC proteins, binds to complete the LRP signalosome.21 Thus the activity of GSK-3β is blocked and β-catenin accumulates in the cytoplasm.22 β-catenin is then able to translocate into the nucleus where it associates with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors that subsequently activate expression of a number of genes (in part by displacing Groucho and HDAC) including those involved in epithelial-to-mesenchymal transition and cell proliferation such as c-myc, c-jun, cyclin D1 and CD44 (Fig. 1). In the absence of Wnt binding, the protein ‘destruction complex’ of GSK-3β along with CKIα, APC and Axin, phosphorylates β-catenin, targeting it for proteolytic degradation (Fig. 1). However, Lee et al.,23 recently demonstrated that Dvl overexpression is itself enough to promote transcriptional activation of TCF/LEF-responsive genes, suggesting that if the concentration of Dvl is high enough to support self-association near the plasma membrane, LRP5/6 can assemble and form the LRP signalosome even in the absence of a Wnt ligand.

As part of the normal regulation of the Wnt pathway to maintain controlled growth, several secreted Wnt antagonists prevent Wnt from binding FRZ [such as Wnt inhibitory factor-1 (WIF1) and secreted frizzled-related proteins (SFRPs)], or to LRP5/6 [DICKKOPF proteins (DKKs)]. These inhibitors prevent the downstream signal transduction and thus help to prevent TCF/LEF-responsive gene expression (reviewed in refs. 24 and 25). The observation that β-catenin expression and autocrine Wnt signaling in breast cancer cells are responsive to exogenous DKK1 and SFRP1 supports the notion that loss of Wnt antagonist expression is an important mechanism in tumorigenesis.18

Regulation of Wnt Pathway Components through Methylation

Aberrant DNA methylation is common in human neoplasia, and hypermethylation of gene-promoter regions is the predominant mechanism leading to loss of gene expression.1,26 Hypermethylation of numerous genes has been documented in various tumor types. Recently, promoter methylation of genes involved in the Wnt-β-catenin pathway was observed in breast cancer (Table 1).12,27–30 This is not surprising since it is known that β-catenin is localized in the nucleus in breast cancer,11,31,32 and activating genetic mutations in Wnt pathway members are rare.

Table 1.

Known and potential methylation-regulated Wnt pathway genes in breast cancer

Gene Methylation confirmed Reference
WIF1 Yes 27, 30
SFRP1–5 Yes 12, 28, 34, 35
APC Yes 7, 9, 29, 36
DKK1 Yes 28, 43
DKK3 No 44, 45, 46
Hint1 No 48, 49
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Wnt inhibitory factor-1 (WIF1, Fig. 1) is a conserved Wnt-binding protein that is downregulated in many cancer types, including prostate, breast, lung, melanoma, colorectal and bladder, and thus is a potential tumor suppressor.30 Ai et al., examined DNA methylation of the WIF1 promoter in both human breast cancer cell lines and in human breast tumors.27 In tumor tissue, methylation-specific PCR (MSP, reviewed in ref. 33) revealed that 16 of 24 tumors (67%) displayed WIF1 promoter hypermethylation. These results agree with those of Wissmann et al., who determined that WIF1 expression was downregulated in 63% (17/27) of invasive ductal carcinomas and in 50% (4/8) of non-ductal invasive breast carcinomas.30 Interestingly, gene knockout studies of DNMT1 and DNMT3β result in significant re-expression of the WIF1 whereas single disruption of either of these genes alone resulted in only minor re-expression of WIF1.27 The re-expression was correlated with a 90% decrease in WIF1 methylation. These results indicate that DNMT1 and DNMT3β work in a cooperative fashion to methylate the WIF1 promoter.27 In summary, these findings suggest that epigenetic silencing of the WIF1 gene is a common event and contributing factor in breast tumorigenesis.

Several frizzled-related proteins (SFRP1–5) antagonize Wnt signaling, and SFRP1 gene is silenced in a number of different cancers, including breast cancer.34 One study12 evaluated 43 tumor and matching normal DNA samples for loss of heterozygosity at the SFRP1 locus and found 11 tumors had allelic loss in chromosome 8p, a site frequently altered in several tumors types. No genetic changes were detected on the remaining gene copy, suggesting SFRP1 may be silenced through methylation. SFRP1 promoter methylation was detected in eight breast cancer cell lines via MSP. More importantly, 79 of 130 (61%) primary breast cancers evaluated also displayed SFRP1 promoter methylation. Kaplan-Meier analysis showed SFRP1 gene hypermethylation was associated with a shorter overall survival of patients with invasive breast cancer, suggesting SFRP1 methylation status may be of predictive value. Another study found similar results despite using fewer samples.35

Recently, Suzuki et al., examined the methylation status of SFRPs 1, 2 and 5 in multiple breast cancer cell lines and patient samples.28 Gene expression analysis using RT-PCR and MSP confirmed that SFPR1 is downregulated and was methylated in 64% of breast cancer cell lines, and they reported for the first time that SFRP2 is methylated in 100% of the cells lines while SFRP5 is methylated in 90% of the breast cancer cell lines examined. As expected, gene expression was restored after treatment with DNMT inhibitors. Analysis of 78 breast tumor samples revealed methylation frequencies of 40, 77 and 71% for SFRP1, 2 and 5, respectively.

These data demonstrate that loss of various antagonists of the Wnt ligand through promoter methylation leads to overactivation of the Wnt signaling pathway, promoting tumorigenesis in human mammary tissues.12 Additional evidence implicating the Wnt pathway comes from the observation that β-catenin (and other downstream targets such as cyclin D1 and LEF) are upregulated in over 40% of breast cancers.10,11 Other Wnt pathway components are also modified through DNA methylation. For example, several studies showed that the APC promoter is methylated in ~35–50% of breast cancer tumors and cell lines.7,9,29,36 and that APC expression is correlated with methylation status. Though APC mutations occur in less than 20% of primary tumor isolates,6,8 LOH is detected in ~25% of breast tumors.9,37,38 In addition, the APC promoter is also regulated by methylation in prostate cancer.39 Loss of APC favors β-catenin accumulation and TCF/LEF-induced transcription. Wnt-independent accumulation of β-catenin might also be promoted by methylation of CDHI, E-cadherin. Membrane-anchored E-cadherin binds β-catenin in the cytoplasm, and thus its downregulated expression permits cytoplasmic β-catenin accumulation.40 However, data from Sarrio et al., unexpectedly found no increase in β-catenin in lobular breast carcinomas containing methylation-silenced CDHI.9 Thus epigenetic silencing of E-cadherin in breast cancer may lead exclusively to other consequences such as epithelialto-mesenchymal transition and increased invasiveness.41

Other Wnt pathway negative regulators are also candidates for epigenetic gene silencing in cancer. The expression of DKK1, which blocks Wnt signals by targeting LRP5/6 for degradation, is silenced by methylation in colon cancer42 and is repressed in some breast cancer cell lines.28,43 One study found that c-Myc expression results in silencing of DKK1, presumably through transcriptional repression mediated by promoter binding by Myc as determined by ChIP assays.43 However, the authors have not ruled out the contribution of methylation-induced silencing of DKK1,43 and recently DKK1 was found to be methylated in 27% and 19% of breast cancer cell lines and patient samples, respectively.28 DKK3, which blocks Wnt7a but not LRP5/6, is downregulated through promoter methylation in acute lymphoblastic leukemia, multiple myeloma and lung cancer.44–46 Hint1/PKC1 (histidine triad nucleotide-binding protein 1/protein kinase C inhibitor) represses TCF-β-catenin transcription activity and, unless silenced, should block Wnt signal propagation.47 While the Hint1 promoter is methylated in colon and non-small cell lung cancers,48,49 no studies containing rigorous analysis of methylation-induced silencing of these genes in breast cancer have been published.

However, silencing some genes that negatively regulate growth-promoting pathways may be lethal. For example ICAT, or inhibitor of β-catenin and TCF, blocks the interaction of β-catenin and TCF and thus stops the Wnt signal transduction.50 Loss of ICAT expression leads to developmental defects of the kidney,51 and to date there are no published studies reporting epigenetic regulation of ICAT in cancer.

Cancer Stem Cells and Wnt

Recent evidence suggests that cancer stem cells (CSCs) mediate solid tumor initiation and progression (reviewed in ref. 52). The CSC constitutes only a minor subpopulation of cells within the tumor, but can self-renew and also give rise to differentiated tumor cells. In contrast the bulk of the tumor is composed of highly differentiated cells with limited proliferative potential. Wnt, together with Notch and Hedgehog pathways, are involved in stem cell self-renewal in hematopoietic, leukemia and some solid tumors (reviewed in ref. 53 and 54). The Wnt pathway is also upregulated in prostate tumor-initiating cells.55 While it is known that Wnt signaling is important for normal stem cell self-renewal and mammary gland development,56 it remains to be determined if and how Wnt signaling and its epigenetic regulation in the CSC contribute to breast carcinogenesis.

In summary, acquisition of breast and numerous other cancers are modulated in part through epigenetic silencing of genes that negatively regulate cell proliferation pathways. Thus, the processes of gene hypermethylation and histone hypoacetylation are therapeutically attractive targets because these non-genetic alterations are reversible.2 However, the inherent cytotoxicity of DNA methyltransferase inhibitors such as decitabine [5-aza-2’-deoxycytidine (5-aza-dC)] needs to be reduced if they are to have a prominent role in cancer therapy.57 Perhaps second-generation inhibitors, those that single-out and act directly and specifically on DNMTs and/or HDAC enzymes, or even drugs targeting cancer stem cells may prove to be successful in managing these diseases. In addition, Schlange et al.,58 demonstrated that interference with autocrine Wnt signaling decreases survival and proliferation of breast cancer cells. These data support the idea that targeted inhibition of Wnt pathways components may be an effective means to control breast cancer.

Acknowledgements

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute.

Abbreviations:

DNMT

DNA methyltransferase

HAT

histone acetyl transferase

HDAC

histone deacetylase

FRZ

Frizzled

LRP

low density lipoprotein receptor-related protein

CK1

casein kinase 1

GSK-3β

glycogen synthase kinase 3β

APC

adenomatous polyposis coli

Dvl

Disheveled

TCF/LEF

T-cell factor/lymphoid enhancer factor

WIF1

Wnt inhibitory factor 1

SFRPs

secreted frizzled-related proteins

DKKs

DICKKOPF proteins

MSP

methylation-specific PCR

Hint1/PKC1

histidine triad-nucleotide binding protein/protein kinase C inhibitor

ICAT

inhibitor of β-catenin

CSC

cancer stem cell

Footnotes

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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