Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer

Abstract

Although mutation of APC or CTNNB1 (b-catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled-related protein (SFRP) and Dickkopf (DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines (SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours (SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines (3 out of 11, 27%) and primary tumours (15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a b-catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T-cell factor/lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis. British Journal of Cancer (2008) Wnt ligands are secreted proteins that bind to transmembrane receptors in the Frizzled (Fz) family. During normal developmental processes, the resultant Wnt signalling plays essential roles in the regulation of cell proliferation, patterning and fate determination (Cadigan and Nusse, 1997). The binding of Wnt to Fz leads to dephosphorylation and stabilisation of b-catenin, enabling it to be translocated into the nucleus, where it interacts with members of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family of transcription factors to stimulate the expression of target genes. This signalling pathway is strongly implicated in tumorigenesis; indeed, the first mammalian Wnt isoform was identified based on its ability to promote mouse mammary tumorigenesis (Polakis, 2000). In addition, aberrant nuclear and cytoplasmic localisation of b-catenin is frequently observed in human breast cancer (Lin et al, 2000;Ryo et al, 2001;Chung et al, 2004). In contrast to colorectal cancer (CRC), however, mutation of APC, AXIN or CTNNB1 (b-catenin) is rare in breast cancer, indicating that other mechanisms are responsible for the activation of b-catenin. These mechanisms could include increased expression of Wnt ligand (Huguet et al, 1994;Dale et al, 1996;Bui et al, 1997) and/or the loss of Wnt antagonists.Several classes of secreted Wnt antagonists are known, including the Cerberus, Wnt inhibitory factor 1, secreted frizzled-related protein (SFRP) and the Dickkopf (DKK) families (Kawano and Kypta, 2003). The SFRP family is comprised of five secreted glycopr...

Prodigiosin inhibits Wnt/β-catenin signaling and exerts anticancer activity in breast cancer cells

“…Wnt ligands and FZD receptors are expressed in human breast cancer cell lines and primary tumors (30)(31)(32)(33). Multiple negative regulators of Wnt/β-catenin signaling, such as SFRP1, SFRP2, SFRP5, WIF1, DKK1, and DKK3, are down-regulated in many breast tumors (34)(35)(36)(37)(38)(39). Although Wnt/β-catenin pathway mutations are rarely detected in breast tumors, elevated levels of nuclear and/or cytoplasmic β-catenin are frequently found and are associated with poor prognosis (40).…”

Resistance or sensitivity of Wilms’ tumor to anti-FZD7 antibody highlights the Wnt pathway as a possible therapeutic target

“…In addition, WTX, which seems to act as a negative regulator of b-catenin, promoting its ubiquitination and degradation (Major et al, 2007) is mutated in WT (Schedl, 2007). Thus, deregulated Wnt/b-catenin signaling with accumulation of b-catenin in WT has been related to genetic events, while alternative mechanisms such as increased expression of Wnt ligands and epigenetic inactivation of Wnt pathway antagonists SFRPs and DKK1, reported in other tumors (Giles et al, 2003;Moon et al, 2004;Katoh and Katoh, 2007;Nojima et al, 2007;Jost et al, 2008;Suzuki et al, 2008), have not been implicated in WT. We hypothesized that in FZD7-resistant WT, activation of b-catenin is likely to be independent on the receptor's action, in contrast to down-stream dependence on receptor activation in the FZD7-senstive tumors.…”

“…Both sFRP1 and DKK1 are known to be epigenetically modified in various types of cancer (Nojima et al, 2007;Jost et al, 2008;Suzuki et al, 2008). Nevertheless, epigenetic regulation of sFRP1 and DKK1 has yet to be demonstrated in WT.…”

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

“…The sFRPs interact with Wnts via the Wnt-binding cysteine rich domain of the Frizzleds and can activate or antagonize Wnt signaling depending on their concentration (71,72). Epigenetic silencing of sFRP promoters by hypermethylation is seen in colorectal and breast cancers (66,69). Wnt inhibitory factor-1, another secreted Wnt inhibitor, is silenced in several tumor types (73)(74)(75).…”

“…There are multiple secreted Wnt antagonists that may normally dampen Wnt signaling but are epigenetically silenced in various cancers (66,69). Most of the reported antagonists function by preventing the interaction of Wnts with their receptors LRP5/6 or Frizzled.…”