Dickkopf-4 is frequently down-regulated and inhibits growth of colorectal cancer cells

Abstract

Like Dickkopf-1 (DKK1), DKK4 is a target of β-catenin/Tcf-4 in colorectal cancer. However, as a negative regulator of Wnt signalling its function in colorectal cancer cells is not well understood. We report that DKK4 is frequently down-regulated in colorectal cancer cell lines with deregulated β-catenin/Tcf-4 and in primary colorectal cancers. Exposure of cancer cells to DKK4 strongly inhibits basal β-catenin/Tcf-4 signalling activity, cancer cell growth and cell cycle progression. Therefore, loss of this negative feed-back loop provides Wnt factor expressing cancer cells with a growth advantage. Our data demonstrate that DKK4 is an important negative regulator of colon cancer cell growth.

Introduction

Wnt/β-catenin signalling plays essential roles in embryogenesis, tissue homeostasis and tumor development [1], [2]. Physiological signalling is initiated by binding of soluble Wnt ligands to members of the Frizzled family of seven-pass transmembrane receptors and LRP5/6 co-receptors. This in turn leads to inhibition of β-catenin phosphorylation by a multi protein complex consisting of CKIα, GSK-3β, Dishevelled, APC, and Axin, and consequently to the inhibition of β-catenin degradation by the ubiquitin/proteosome pathway [1]. β-catenin then translocates to the nucleus, binds to Tcf/LEF factors and orchestrates the transcription of multiple target genes. The coordinated regulation of this canonical Wnt pathway is essential for differentiation of stem cells and homeostasis of tissues including intestinal epithelium, skin, muscle, and hematopoetic cells [3]. Two groups of extracellular Wnt antagonists, the secreted Frizzled-related proteins (sFRPs), and the members of the Dickkopf (DKK) family, act to fine-tune the spatiotemporal patterns of Wnt activity [4]. sFRPs inhibit Wnt signalling by sequestering Wnts. Binding of Dickkopf proteins to the Wnt co-receptor LRP5/6 causes endocytosis of the complex and consecutively inhibition of the recruitment of LRP5/6 to Frizzled receptors which is essential for initiation of Wnt signalling at the cell membrane [5].

Constitutively active Wnt signalling is causally involved in the genesis of various cancers [3], [6]. Most frequently, deregulation of Wnt/β-catenin signalling is found in colorectal cancer due to mutation of APC. This leads to cytoplasmic stabilization of β-catenin and in turn results in the activation of β-catenin/Tcf mediated transcription. In a widely accepted linear view of constitutive activation of Wnt/β-catenin signalling in cancer, additional activating signals are thought to be ineffective and redundant [7]. However, several lines of evidence support a view in which activating and inhibiting signals act simultaneously on different levels of the pathway to modulate Wnt signalling in cancer cells [3]. Additionally, mutant APC retains some residual activity to regulate β-catenin [8], and thus activation of Wnt signalling at the membrane level in addition to APC mutation would allow for further growth advantage of cancer cells. In this model, extracellular Wnt factors might act as a second force to stabilize β-catenin [9]. On this background, silencing of the expression of sFRPs and DKK family members provides a growth advantage for cancer cells [10], [11], [12].

Here, we report that DKK4, which like DKK1 is a target gene of β-catenin/Tcf-4, and would therefore be expected to be strongly expressed in colorectal cancers carrying mutations of APC or β-catenin, is frequently down-regulated in colorectal cancer cell lines and primary tumors. We find that DKK4 acts as a potent inhibitor of Wnt-factor induced Tcf-dependent signalling. Ectopic re-expression of DKK4 or treatment of colorectal cancer cells with recombinant DKK4 resulted in inhibition of cell growth by attenuation of cell cycle progression. We find that down-regulation of DKK4 expression is caused by histone deacetylation. Our data demonstrate that colorectal cancer cells select against the expression of DKK4. Due to abolishment of the negative feed-back loop provided by DKK4 colorectal cancer cells gain further growth advantage. Therefore, DKK4 is a new tumor suppressor gene candidate in colorectal cancer.