Complex interplay between β-catenin signalling and Notch effectors in intestinal tumorigenesis

Abstract

Aims: The activation of β-catenin signalling is a key step in intestinal tumorigenesis. Interplay between the β-catenin and Notch pathways during tumorigenesis has been reported, but the mechanisms involved and the role of Notch remain unclear.

Methods: Notch status was analysed by studying expression of the Notch effector Hes1 and Notch ligands/receptors in human colorectal cancer (CRC) and mouse models of Apc mutation. A genetic approach was used, deleting the Apc and RBP-J or Atoh1 genes in murine intestine. CRC cell lines were used to analyse the control of Hes1 and Atoh1 by β-catenin signalling.

Results: Notch signalling was found to be activated downstream from β-catenin. It was rapidly induced and maintained throughout tumorigenesis. Hes1 induction was mediated by β-catenin and resulted from both the induction of the Notch ligand/receptor and Notch-independent control of the Hes1 promoter by β-catenin. Surprisingly, the strong phenotype of unrestricted proliferation and impaired differentiation induced by acute Apc deletion in the intestine was not rescued by conditional Notch inactivation. Hyperactivation of β-catenin signalling overrode the forced differention induced by Notch inhibition, through the downregulation of Atoh1, a key secretory determinant factor downstream of Notch. This process involves glycogen synthase kinase 3 β (GSK3β) and proteasome-mediated degradation. The restoration of Atoh1 expression in CRC cell lines displaying β-catenin activation was sufficient to increase goblet cell differentiation, whereas genetic ablation of Atoh1 greatly increased tumour formation in Apc mutant mice.

Conclusion: Notch signalling is a downstream target of β-catenin hyperactivation in intestinal tumorigenesis. However, its inhibition had no tumour suppressor effect in the context of acute β-catenin activation probably due to the downregulation of Atoh1. This finding calls into question the use of γ-secretase inhibitors for the treatment of CRC and suggests that the restoration of Atoh1 expression in CRC should be considered as a therapeutic approach.

Figure 1

Induction of Hes1 expression in human and murine intestinal carcinogenesis. Analysis of Hes1 mRNA levels by quantitative reverse transcription–PCR (RT–PCR) in human colorectal cancer (CRC) and in normal tissue (A). Immunostaining for Hes1 and β-catenin in human CRC and in normal tissue (B). Scale bars, 200 μm. Analysis of Hes1 expression by quantitative RT–PCR (C) and immunohistochemistry (D) in normal tissue and adenomas from APC^+/− mice.

Figure 2

Induction of Notch receptors and ligands in human and murine intestinal carcinogenesis. Fold change in mRNA levels for the Notch receptors and ligands, as measured by quantitative reverse transcription–PCR (RT–PCR), in comparisons of human colorectal carcinomas with normal tissue (A). In situ hybridisation of Notch2 and Delta4 in human normal colon and in colorectal cancer (B). Quantitative RT–PCR analyses of mRNA levels for the Notch receptors and ligands in normal tissues and adenomas from APC^+/− mice (C).

Figure 3

Hes1 expression is correlated with Wnt/β-catenin signalling. Quantitative reverse transcription–PCR (RT–PCR) analysis of Hes1 mRNA levels in control and APC^−/− mice 5 days after tamoxifen injection (A). Immunoblot analyses of Hes1 levels in three different samples from control and APC^−/− mice (day 5) (B), and Hes1 and β-catenin levels in SW480 cells after transfection with two small interfering RNAs (siRNAs) against β-catenin or scramble siRNA (D). β-Actin levels were used for normalisation. Immunohistochemistry studies of Hes1 and β-catenin in control and APC^−/− mice (day five) (C). Scale bars, 50 μm.

Figure 4

The Hes1 promoter is regulated by Wnt/β-catenin signalling through conserved Tcf motifs. Quantitative reverse transcription–PCR (RT–PCR) analysis of mRNA levels for the Notch receptors and ligands in control and APC^−/− mice (day 5) (A). Conserved Tcf-binding element motifs identified by Genomatix software in the proximal region of the Hes1 promoter of several species. The putative Tcf- and RBP-J-binding sites are represented by blue and purple boxes, respectively. Maps of the constructs used to study the transcriptional activity of the Hes1 promoter in SW480 cells. Different fragments of regulatory sequences flanking the human Hes1 promoter (Hes-2.5, Hes-0.4 and Hes-mutRBP constructs) were fused with the luciferase gene. The Hes-mutRBP construct contains only the Tcf sites, the RBP-J-binding sites having been mutated (B). All the constructs were used to transfect SW480 cells together with the Notch1 intracellular domain (NICD), the dominant-negative form of Tcf4 (ΔNTCF4) or empty vector (control) (C–E).

Figure 5

Loss of RBP-J does not alter the cell differentiation defects of Apc-deficient cells. Kaplan–Meier survival analysis of control (purple), APC^−/− (green) and APC^−/−RBP^−/− mice (red) (injections are indicated by an arrow) (A). Representative H&E-stained sections of control, APC^−/− and APC^−/−RBP^−/− mice 5 days after tamoxifen (Tam) injection (day 5) (B). PCR of DNA extracted from intestines of control and APC^−/−RBP^−/− mice showing the excision of the floxed RBP-J alleles (day 5) (C). Quantitative reverse transcription–PCR (RT–PCR) analysis of Atoh1, p27^kip1 and p57^kip2 mRNA levels in control, APC^−/− and APC^−/−RBP^−/− mice (day 5) (D). Immunoblot analyses of Hes1 levels in three different samples from control, APC^−/−, RBP^−/− and APC^−/−RBP^−/− mice (day 5) (E). β-Actin levels were used for normalisation. Alcian blue staining for control, APC^−/−, RBP^−/− and APC^−/−RBP^−/− mice (F). Quantitative RT–PCR analysis of Muc2, Gob4 and Gob5 mRNA levels in control, APC^−/−, RBP^−/− and APC^−/−RBP^−/− mice (day 5) (G). For statistical analyses mutant mice were compared with control mice. Scale bars, 100 μm.

Figure 6

Loss of RBP-J does not affect the increase in cell proliferation in Apc-deficient cells. Analyses of cell proliferation by immunohistochemistry (A). Bromodeoxyuridine (BrdU), phosphorylated histone H3 (pH3) and Ki-67 staining was performed on control, APC^−/− and APC^−/−RBP^−/− mice, 5 days after tamoxifen (Tam) injection. Quantification of BrdU- and pH3-positive cells per crypt in control, APC^−/− and APC^−/−RBP^−/− mice (B). Scale bars, 100 μm. Quantitative reverse transcription–PCR (RT–PCR) analyses of mRNA for various cell cycle components in control, APC^−/− and APC^−/−RBP^−/− mice (day 5). For statistical analyses, mutant mice were compared with control mice (C).

Figure 7

Loss of RBP-J does not affect the stem cell amplification or apoptosis induced by Apc deficiency. Identification of apoptotic cells in control, APC^−/− and APC^−/−RBP^−/− mice, 5 days after tamoxifen (Tam) injection, by the deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method (A). Scale bars, 100 μm. Quantitative reverse transcription–PCR (RT–PCR) analyses of mRNA levels for stem cell markers in control, APC^−/− and APC^−/−RBP^−/− mice (day 5). For statistical analyses, mutant mice were compared with control mice (B).

Figure 8

β-Catenin signalling controls Atoh1 expression post-transcriptionally. Co-transfections of HEK-293T cells with various amounts of plasmid encoding Atoh1-haemagglutinin (HA) (0, 50 and 150 ng) and an oncogenic mutant form of β-catenin (0 and 500 ng). Atho1 protein levels were analysed by immunoblotting of the HA tag and quantified by Genetool from SynGene software (A). β-Actin levels were used for normalisation. Analyses of Atho1 and Muc2 transcriptional activity in SW480, TC7 and HT-29 cells by transfection with plasmids encoding luciferase under the control of the Atoh1 (Enh-Atoh1-Luc) or Muc2 regulatory sequences (Muc2-Luc). Cells were treated with glycogen synthase kinase 3 β (GSK3β) inhibitor (AR-A014418) for 48 h or with proteasome inhibitor (MG132) for 18 h before the quantification of luciferase activity (B).

Figure 9

Loss of Atoh1 enhances the tumour formation induced by Apc loss. Alcian blue staining and immunostaining for lysosyme in Apc^+/− and Apc^+/−Atoh1^−/− mice (A). Mean number of lesions per animal in the small intestine and colon of Apc^+/− (n=5) and Apc^+/−Atoh1^−/− (n=7) mice (B). H&E staining and immunostaining for β-catenin in Apc^+/−Atoh1^−/− mice (C).