Several recent studies have suggested that the Wnt and Notch pathways cooperate to trigger intestinal tumorigenesis.7 9 12
However, the molecular mechanism involved in this cross-talk remains unclear. We have shown that Notch activation, as reflected by overproduction of the Hes1 and Notch ligands and receptors, is a frequent and early event in Wnt-induced intestinal tumorigenesis, and that this activation is maintained throughout tumorigenesis and downstream from the Wnt/β-catenin cascade. We show that Hes1 overexpression in intestinal cancer results from two complementary processes controlled by β-catenin. One is the β-catenin-mediated upregulation of the ligands and receptors of the Notch pathway, as previously described.9 12 24
However, among all the Notch ligands and receptors tested, only the expression of Jag1 appears directly affected by β-catenin activation. The upregulation of the other members appears to be an indirect consequence of the activation of the β-catenin signalling. The second process is a direct increase in Hes1 promoter activity mediated by β-catenin/Tcf signalling. Indeed, although Hes1 is a genuine Notch effector, it may also be activated by other signalling pathways, such as those of Wnt, Sonic Hedgehog, IκBα and Ras/MAPK.25–28
Altogether, we and others showed a complex integration of the Wnt and Notch pathways in intestinal epithelial cells. During intestine development, it has been shown that the effects of Notch signalling on intestinal cell proliferation are dependent on Wnt. This epistatic study indicates that Notch acts upstream of Wnt. However, in adult intestine, during Apc-driven tumorigenesis, our data indicate that activation of Notch acts downstream of Wnt. This statement is in accordance with other published data.12 29
Altogether, it appears that the interaction between the Wnt and the Notch pathways is complex and appears to be context dependent.
We analysed the role of Notch in tumorigenesis, by assessing the effect of blocking Notch signalling in cells in which β-catenin was deregulated, mimicking the early stage of tumorigenesis. We used conditional targeting methods to delete the Apc and RBP-J genes simultaneously in the intestinal epithelium of adult mice. As the treatment of ApcMin
adenomas with γ-secretase inhibitors led to the conversion of some adenoma cells to goblet cells,7
we expected to alter the initiation of tumour development induced by acute Apc loss. However, Notch inhibition did not rescue the severe phenotype caused by Apc loss in the intestine. No commitment to goblet cell lineage differentiation was observed, and unrestricted proliferation, with enlarged, highly proliferative crypts and stem cell amplification was seen in all cases, regardless of Notch status. Based on published data, one possible explanation is that acute Wnt/β-catenin signalling could modulate expression of Atoh1, a downstream component of Notch.18 19
Using CRC cell lines, we showed that endogenous Atoh1 protein levels were downregulated by Wnt/β-catenin signalling. The stabilisation of Atoh1 protein by GSK3β and proteasome inhibitors allowed the expression of the goblet cell marker. Conversely, we showed that genetic ablation of Atoh1 dramatically accelerated in 3 weeks the emergence of Apc-mediated adenomas throughout the whole small and large intestine (results of this study,22
). Consistent with the tumour suppressor role of Atoh1 in CRCs, recent studies have reported the epigenetic and genetic silencing of the Atoh1 gene in human CRC.22
These results highlight a critical role for Atoh1 in the oncogenic effect of β-catenin signalling in intestinal tumorigenesis.
Our data contrast with those of a study reporting the conversion of adenoma cells into goblet cells after the treatment of ApcMin
mice with γ-secretase inhibitors.7
However, conversion to goblet cells was nonetheless relatively rare. Almost 50% of adenomas showed no conversion of tumours cell and only 28% displayed the conversion of 1–10% of tumour cells into goblet cells.7
There may be several reasons for the discrepancies between these two studies. As Notch inactivation was achieved by deletion of the RBP gene or with γ-secretase inhibitors, we cannot exclude the possibility of an RBP-independent effect of Notch.30
However, it is more likely that the different effects of Notch inhibition on the Apc-mediated phenotype are linked to different β-catenin activation thresholds. Indeed, a dose-dependent effect of β-catenin has already been described.31 32
Based on the expression profile of the β-catenin target gene c-myc that we have previously described,3 10
it appears that the level of activation of Wnt/β-catenin signalling differs between the two mouse models. Strong and homogeneous β-catenin activation probably leads to Atoh1 destabilisation in the presence of a conditional homozygous deletion of Apc, whereas the heterogeneous β-catenin activation in ApcMin
mice may allow Notch inhibition by maintaining Atoh1 levels above a threshold sufficient to induce goblet differentiation, in a subset of cells. A recent study showed that the complete deletion of Atoh1 abolished the effects of γ-secretase inhibitors on cancerous intestinal cells.33
Our data highlight the complex interplay between the Wnt and Notch signalling pathways during intestinal tumorigenesis. Two main Notch effectors are controlled in opposite ways by the Wnt/β-catenin pathway. Hes1 is transcriptionally upregulated, whereas Atoh1 is post-transcriptionally downregulated. This downregulation is critical to the oncogenic outcome mediated by β-catenin, because it prevents the differentiation of cancer cells. Our findings have important clinical implications, as they call into question the utility of γ-secretase inhibitors as a treatment for CRC and suggest that the stabilisation of Atoh1 may be of therapeutic importance.