Is there more to Wnt signalling in breast cancer than stabilisation of β-catenin?

Increased Wnt signalling has been implicated in the aetiology of many different human cancers, including breast cancers. In most cases, Wnt signalling is thought to drive tumourigenesis through the stabilisation of cytosolic β-catenin and the subsequent changes in the expression of T-cell factor (TCF)-dependent genes. However, this is not necessarily the only mechanism, as Wnt proteins can signal through a number of different intracellular signalling pathways. The ongoing work from Nancy Hynes' laboratory continues to highlight this latter possibility

Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation

The low density lipoprotein receptor-related protein-6 (LRP6) is an essential co-receptor for canonical Wnt signaling. Dickkopf 1 (Dkk1), a major secreted Wnt signaling antagonist, binds to LRP6 with high affinity and prevents the Frizzled-Wnt-LRP6 complex formation in response to Wnts. Previous studies have demonstrated that Dkk1 promotes LRP6 internalization and degradation when it forms a ternary complex with the cell surface receptor Kremen.In the present study, we found that transfected Dkk1 induces LRP6 accumulation while inhibiting Wnt/LRP6 signaling. Treatment with Dkk1-conditioned medium or recombinant Dkk1 protein stabilized LRP6 with a prolonged half-life and induces LRP6 accumulation both at the cell surface and in endosomes. We also demonstrated that Kremen2 co-expression abrogated the effect of Dkk1 on LRP6 accumulation, indicating that the effect of Kremen2 is dominant over Dkk1 regulation of LRP6. Furthermore, we found that Wnt3A treatment induces LRP6 down-regulation, an effect paralleled with a Wnt/LRP6 signaling decay, and that Dkk1 treatment blocked Wnt3A-induced LRP6 down-regulation. Finally, we found that LRP6 turnover was blocked by an inhibitor of caveolae-mediated endocytosis.Our results reveal a novel role for Dkk1 in preventing Wnt ligand-induced LRP6 down-regulation and contribute significantly to our understanding of Dkk1 function in Wnt/LRP6 signaling

Raphe-mediated signals control the hippocampal response to SRI antidepressants via miR-16

Serotonin reuptake inhibitor (SRI) antidepressants such as fluoxetine (Prozac), promote hippocampal neurogenesis. They also increase the levels of the bcl-2 protein, whose overexpression in transgenic mice enhances adult hippocampal neurogenesis. However, the mechanisms underlying SRI-mediated neurogenesis are unclear. Recently, we identified the microRNA miR-16 as an important effector of SRI antidepressant action in serotonergic raphe and noradrenergic locus coeruleus (LC). We show here that miR-16 mediates adult neurogenesis in the mouse hippocampus. Fluoxetine, acting on serotonergic raphe neurons, decreases the amount of miR-16 in the hippocampus, which in turn increases the levels of the serotonin transporter (SERT), the target of SRI, and that of bcl-2 and the number of cells positive for Doublecortin, a marker of neuronal maturation. Neutralization of miR-16 in the hippocampus further exerts an antidepressant-like effect in behavioral tests. The fluoxetine-induced hippocampal response is relayed, in part, by the neurotrophic factor S100β, secreted by raphe and acting via the LC. Fluoxetine-exposed serotonergic neurons also secrete brain-derived neurotrophic factor, Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2. These molecules are unable to mimic on their own the action of fluoxetine and we show that they act synergistically to regulate miR-16 at the hippocampus. Of note, these signaling molecules are increased in the cerebrospinal fluid of depressed patients upon fluoxetine treatment. Thus, our results demonstrate that miR-16 mediates the action of fluoxetine by acting as a micromanager of hippocampal neurogenesis. They further clarify the signals and the pathways involved in the hippocampal response to fluoxetine, which may help refine therapeutic strategies to alleviate depressive disorders

A Cryptic Frizzled Module in Cell Surface Collagen 18 Inhibits Wnt/β−Catenin Signaling

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (Mr = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate

A System-Wide Investigation of the Dynamics of Wnt Signaling Reveals Novel Phases of Transcriptional Regulation

Aberrant Wnt signaling has been implicated in a wide variety of cancers and many components of the Wnt signaling network have now been identified. Much less is known, however, about how these proteins are coordinately regulated. Here, a broad, quantitative, and dynamic study of Wnt3a-mediated stimulation of HEK 293 cells revealed two phases of transcriptional regulation: an early phase in which signaling antagonists were downregulated, providing positive feedback, and a later phase in which many of these same antagonists were upregulated, attenuating signaling. The dynamic expression profiles of several response genes, including MYC and CTBP1, correlated significantly with proliferation and migration (P<0.05). Additionally, their levels tracked with the tumorigenicity of colon cancer cell lines and they were significantly overexpressed in colorectal adenocarcinomas (P<0.05). Our data highlight CtBP1 as a transcription factor that contributes to positive feedback during the early phases of Wnt signaling and serves as a novel marker for colorectal cancer progression

DNA methylation status of REIC/Dkk-3 gene in human malignancies

The REIC (reduced expression in immortalized cells)/Dkk-3 is down-regulated in various cancers and considered to be a tumor suppressor gene. REIC/Dkk-3 mRNA has two isoforms (type-a,b). REIC type-a mRNA has shown to be a major transcript in various cancer cells, and its promoter activity was much stronger than that of type-b. In this study, we examined the methylation status of REIC/Dkk-3 type-a in a broad range of human malignancies. We examined REIC/Dkk-3 type-a methylation in breast cancers, non-small-cell lung cancers, gastric cancers, colorectal cancers, and malignant pleural mesotheliomas using a quantitative combined bisulfite restriction analysis assay and bisulfate sequencing. REIC/Dkk-3 type-a and type-b expression was examined using reverse transcriptional PCR. The relationships between the methylation and clinicopathological factors were analyzed. The rate of REIC/Dkk-3 type-a methylation ranged from 26.2 to 50.0% in the various primary tumors that were examined. REIC/Dkk-3 type-a methylation in breast cancer cells was significantly heavier than that in the other cell lines that we tested. REIC/Dkk-3 type-a methylation was inversely correlated with REIC/Dkk-3 type-a expression. There was a correlation between REIC/Dkk-3 type-a and type-b mRNA expression. REIC/Dkk-3 type-a expression was restored in MDA-MB-231 cells using 5-aza-2'-deoxycytidine treatment. We found that estrogen receptor-positive breast cancers were significantly more common among the methylated group than among the non-methylated group. REIC/Dkk-3 type-a methylation was frequently detected in a broad range of cancers and appeared to play a key role in silencing REIC/Dkk-3 type-a expression in these malignancies

Dickkopf1 - A New Player in Modelling the Wnt Pathway

The Wnt signaling pathway transducing the stabilization of β-catenin is essential for metazoan embryo development and is misregulated in many diseases such as cancers. In recent years models have been proposed for the Wnt signaling pathway during the segmentation process in developing embryos. Many of these include negative feedback loops where Axin2 plays a key role. However, Axin2 null mice show no segmentation phenotype. We therefore propose a new model where the negative feedback involves Dkk1 rather than Axin2. We show that this model can exhibit the same type of oscillations as the previous models with Axin2 and as observed in experiments. We show that a spatial Wnt gradient can consistently convert this temporal periodicity into the spatial periodicity of somites, provided the oscillations in new cells arising in the presomitic mesoderm are synchronized with the oscillations of older cells. We further investigate the hypothesis that a change in the Wnt level in the tail bud during the later stages of somitogenesis can lengthen the time period of the oscillations and hence the size and separation of the later somites

The Wnt pathway regulator DKK1 is preferentially expressed in hormone-resistant breast tumours and in some common cancer types

In addition to new tumour antigens, new prognostic and diagnostic markers are needed for common cancers. In this study, we report the expression of Dickkopf-1 (DKK1) in multiple common cancers. This constitutes a comprehensive analysis of the DKK1 expression profile. Dickkopf-1 expression was evaluated by classical and quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbant assay for protein determination, in cancer lines and clinical specimens of several cancer origins. For breast cancer, expression was correlated with clinicopathological parameters. Dickkopf-1 expression was confirmed in several cancer cell lines derived from breast and other common cancers. Dickkopf-1 protein secretion was documented in breast, prostate and lung cancer lines, but was negligible in melanoma. Analysis of DKK1 expression in human cancer specimens revealed DKK1 expression in breast (21 out of 73), lung (11 out of 23) and kidney cancers (six out of 20). Interestingly, DKK1 was preferentially expressed in oestrogen and progesterone receptor-negative tumours (ER−/PR−; P=0.005) and in tumours from women with a family history of breast cancer (P=0.024). Importantly, DKK1 protein production was confirmed in multiple breast cancer specimens that were positive by RT–PCR. This work establishes DKK1 as a potential prognostic and diagnostic marker for cohorts of breast cancer patients with poor prognosis. Dickkopf-1 may also become a relevant candidate target for immunotherapy of different cancers

Down-regulation of frizzled-7 expression decreases survival, invasion and metastatic capabilities of colon cancer cells

BackgroundThe canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in colon cancer cells.Methods and resultsIn this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40-50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P&lt;0.005), and overall survival was shorter in those patients with higher FZD7 expression (P&lt;0.001).ConclusionThese data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway

Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis

The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases