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Reorganisation of Wnt-response pathways in colorectal tumorigenesis.

Caldwell GM, Jones CE, Soon Y, Warrack R, Morton DG, Matthews GM - Br. J. Cancer (2008)

Bottom Line: In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment.These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers.These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Sciences, Epithelial Research Group, School of Medicine, The University of Birmingham, Edgbaston, Birmingham B15 2TH, UK. G.M.Caldwell@bham.ac.uk

ABSTRACT
In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

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Induction of NKD1 and FZD3 in cultured cells. (A) Lithium chloride induction of NKD1 mRNA in FHs74Int cells. Confluent cells were treated with lithium chloride and sampled over a 16-h time course. Expression of FZD3 and NKD1 was measured by real-time RT–PCR and the level relative to untreated control cells determined. The inset panel shows qualitative RT–PCR analysis of the induced cells at 0 and 16 h. (B) Wnt3a induction of NKD1 and FZD3 mRNA in HeLa cells. Confluent cells were treated with Wnt3a-conditioned medium and sampled over a 17-h time course. Expression of NKD1 and FZD3 was measured by real-time RT–PCR and the level relative to untreated control cells determined.
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fig4: Induction of NKD1 and FZD3 in cultured cells. (A) Lithium chloride induction of NKD1 mRNA in FHs74Int cells. Confluent cells were treated with lithium chloride and sampled over a 16-h time course. Expression of FZD3 and NKD1 was measured by real-time RT–PCR and the level relative to untreated control cells determined. The inset panel shows qualitative RT–PCR analysis of the induced cells at 0 and 16 h. (B) Wnt3a induction of NKD1 and FZD3 mRNA in HeLa cells. Confluent cells were treated with Wnt3a-conditioned medium and sampled over a 17-h time course. Expression of NKD1 and FZD3 was measured by real-time RT–PCR and the level relative to untreated control cells determined.

Mentions: The correlation between NKD1, FZD3 and FZD6 and β-catenin stabilisation indicates a cell autonomous effect in the tumour epithelium. In the absence of reliable reagents to assess this in histological sections, we attempted to recapitulate these events in vitro. Previous experiments (Yan et al, 2001) demonstrated β-catenin dependence of NKD1 transcription. Stabilisation of β-catenin in cultured cells would determine whether the FZD expression changes are cell autonomous. The epithelial cell line Fhs74Int, which was isolated from normal intestine (Owens et al, 1976) and retains normal APC function, was used for these experiments. We treated confluent cultures with lithium chloride, an inhibitor of glycogen synthase kinase-3β (GSK-3β), to stabilise β-catenin (Klein and Melton, 1996), and measured changes in NKD1 and FZD3. Figure 4A shows that NKD1 levels increased substantially, reaching a peak after 8 h and dropping gradually towards 16 h after induction, while FZD3 levels were essentially unaffected by lithium treatment, indicating that β-catenin stabilisation alone cannot account for the induction of FZD3 observed in adenomas.


Reorganisation of Wnt-response pathways in colorectal tumorigenesis.

Caldwell GM, Jones CE, Soon Y, Warrack R, Morton DG, Matthews GM - Br. J. Cancer (2008)

Induction of NKD1 and FZD3 in cultured cells. (A) Lithium chloride induction of NKD1 mRNA in FHs74Int cells. Confluent cells were treated with lithium chloride and sampled over a 16-h time course. Expression of FZD3 and NKD1 was measured by real-time RT–PCR and the level relative to untreated control cells determined. The inset panel shows qualitative RT–PCR analysis of the induced cells at 0 and 16 h. (B) Wnt3a induction of NKD1 and FZD3 mRNA in HeLa cells. Confluent cells were treated with Wnt3a-conditioned medium and sampled over a 17-h time course. Expression of NKD1 and FZD3 was measured by real-time RT–PCR and the level relative to untreated control cells determined.
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Related In: Results  -  Collection

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fig4: Induction of NKD1 and FZD3 in cultured cells. (A) Lithium chloride induction of NKD1 mRNA in FHs74Int cells. Confluent cells were treated with lithium chloride and sampled over a 16-h time course. Expression of FZD3 and NKD1 was measured by real-time RT–PCR and the level relative to untreated control cells determined. The inset panel shows qualitative RT–PCR analysis of the induced cells at 0 and 16 h. (B) Wnt3a induction of NKD1 and FZD3 mRNA in HeLa cells. Confluent cells were treated with Wnt3a-conditioned medium and sampled over a 17-h time course. Expression of NKD1 and FZD3 was measured by real-time RT–PCR and the level relative to untreated control cells determined.
Mentions: The correlation between NKD1, FZD3 and FZD6 and β-catenin stabilisation indicates a cell autonomous effect in the tumour epithelium. In the absence of reliable reagents to assess this in histological sections, we attempted to recapitulate these events in vitro. Previous experiments (Yan et al, 2001) demonstrated β-catenin dependence of NKD1 transcription. Stabilisation of β-catenin in cultured cells would determine whether the FZD expression changes are cell autonomous. The epithelial cell line Fhs74Int, which was isolated from normal intestine (Owens et al, 1976) and retains normal APC function, was used for these experiments. We treated confluent cultures with lithium chloride, an inhibitor of glycogen synthase kinase-3β (GSK-3β), to stabilise β-catenin (Klein and Melton, 1996), and measured changes in NKD1 and FZD3. Figure 4A shows that NKD1 levels increased substantially, reaching a peak after 8 h and dropping gradually towards 16 h after induction, while FZD3 levels were essentially unaffected by lithium treatment, indicating that β-catenin stabilisation alone cannot account for the induction of FZD3 observed in adenomas.

Bottom Line: In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment.These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers.These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Sciences, Epithelial Research Group, School of Medicine, The University of Birmingham, Edgbaston, Birmingham B15 2TH, UK. G.M.Caldwell@bham.ac.uk

ABSTRACT
In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

Show MeSH
Related in: MedlinePlus