Expression of sFRP-4 and β-catenin in human colorectal carcinoma

Abstract

Alterations to the Wnt signalling pathway occur in the majority of colorectal cancers and result in abnormal accumulation of β-catenin. The secreted frizzled related proteins (sFRPs) are antagonists that bind Wnt and inhibit signalling along this pathway. We investigated expression of the sFRP family member, sFRP-4, and β-catenin in 1044 human colorectal carcinomas using tissue microarrays and immunohistochemistry. Both proteins showed markedly increased expression levels in tumors compared to normal mucosa, but no significant associations with pathological features or with patient outcome. sFRP-4 was co-expressed with β-catenin, p53, and COX-2, while the absence of β-catenin expression was strongly associated with loss of expression of the MLH1 mismatch repair gene. In contrast to other sFRP family members, sFRP-4 expression appears to be upregulated in colorectal carcinoma.

Introduction

Wnt genes encode a large family of secreted proteins that play key roles as intercellular signalling molecules in development. The transduction of Wnt signals between cells proceeds via a complex series of events that include post-translational modification and secretion of Wnts, binding to transmembrane receptors, activation of cytoplasmic effectors and transcriptional regulation of target genes [1]. In adult tissues, Wnt signalling is involved in proliferation, differentiation and apoptotic pathways [2]. Aberrant regulation of the Wnt signalling pathway has therefore been suggested to play a role in tumorigenesis [3] and in particular the development of colorectal cancer [4]. This occurs mainly through mutations in APC but also of the CTNNB1 gene encoding β-catenin [5], [6]. Mutation of these genes leads to ligand-independent Wnt signalling and results in the abnormal accumulation of free β-catenin in the nucleus. This in turn activates transcription factors such as T-cell factor to induce the expression of target genes involved in cell proliferation, including c-myc and cyclin D1.

Secreted Frizzled Related Proteins (sFRPs) are antagonists that bind directly to Wnts and prevent them from binding to their receptors [7]. They contain a cysteine-rich domain with similarity to the ligand-binding domain of the Frizzled transmembrane protein family, but lack the transmembrane component required for anchoring to the plasma membrane [8]. The inhibitory effects of sFRPs on Wnt signalling have been demonstrated in Xenopus embryos as well as in cultured cells [9], [10], while the expression pattern of several sFRPs complement those of specific Wnts, possibly indicating direct antagonism of Wnt function.

Hypermethylation of the promoter region of sFRP genes occurs frequently in colorectal tumors and is associated with the transcriptional silencing of these genes [11], [12]. Tumor cells may gain a selective advantage by shutting down the expression of normally proapoptotic sFRPs [13], [14]. Epigenetic loss of sFRP function occurs early in colorectal cancer progression and may thus lead to constitutive Wnt signalling that complements downstream mutations [12]. Restoration of sFRP function in colorectal cancer cells has been shown to attenuate Wnt signalling even in the presence of downstream mutations [12].

In the present study we investigated the expression of one of the sFRP family members, sFRP-4, together with β-catenin in a large cohort of colorectal tumors by using tissue microarrays (TMA) and immunohistochemistry (IHC). This has allowed us to examine the pattern of expression of these proteins in relation to each other and to various pathological and molecular features including prognosis.