Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer

Abstract

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers 1 . It occurs through mutations mainly of APC and less often of CTNNB1 (encoding β-catenin) 1-3 or AXIN2 (encoding axin-2, also known as conductin) 4 . These mutations allow ligandindependent WNT signaling that culminates in abnormal accumulation of free β-catenin in the nucleus 1-3 . We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzledrelated proteins (SFRPs) in colorectal cancer 5 . SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development 6-10 . Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin

“…As negative controls, we prepared cells expressing either the homologous C18 polypeptide lacking the frizzled module (V2Nter-HCT116) or empty pCDNA3.1 vector (Vector-HCT116, Figures 1a and b). In HCT116 cells, transient expression of V3Nter, SFRP-1 or SFRP-5 induces cell death (Suzuki et al, 2004;Quelard et al, 2008). Consistently, attempts to obtain clones by limiting dilution were unsuccessful, suggesting a negative selection pressure, as shown for SFRP-1 in breast cancer cells (Bafico et al, 2004).…”

“…It has been suggested that SFRPs may function as tumor suppressors in CRC because allelic loss or epigenetic inactivation of SFRP genes contributes to constitutive activation of the Wnt/b-catenin pathway (Caldwell et al, 2004;Suzuki et al, 2004). In this context, we and others have shown that restoring expression of SFRP-1 or SFRP-5 inhibits Wnt/b-catenin stabilization and induces in vitro cell death in human CRC cells (Suzuki et al, 2004;Quelard et al, 2008).…”

“…An increasing body of evidence Suzuki et al, 2004;He et al, 2005) indicates that the mechanism of b-catenin degradation may be different in cancer cells with respect to normal cells (Liu et al, 2002), supporting the notion that b-TrCP-independent pathways could be alternatively involved. Indeed, the ubiquitination of b-catenin by different E3 ligases that can bypass b-catenin phosphorylation occurs in specific contexts.…”

“…Enhanced autocrine Wnt signaling (Bafico et al, 2004) and epigenetic silencing of genes encoding endogenous extracellular Wnt inhibitors (Suzuki et al, 2004) provide a positive selection advantage to cells carrying b-catenin pathway mutations (Barker and Clevers, 2006;Polakis, 2007;MacDonald et al, 2009). In this setting, frizzled receptor activation and enhanced Wnt expression drive positive cancer cell selection (Vider et al, 1996; Smith et al, An SFRP-like frizzled motif blocks tumor growth E Lavergne et al 1999; Dimitriadis et al, 2001;Holcombe et al, 2002;Ueno et al, 2008).…”

“…In this context, we and others have shown that restoring expression of SFRP-1 or SFRP-5 inhibits Wnt/b-catenin stabilization and induces in vitro cell death in human CRC cells (Suzuki et al, 2004;Quelard et al, 2008). In addition to SFRPs, other endogenous molecules carrying frizzled CRDs bind to and inhibit Wnt signaling.…”