In this study we show that ATOH1 is critically required for all effects of Notch/γ-secretase inhibitors on intestinal progenitor cells. Our results suggest a model in which the primary function of Notch in intestinal stem cells is to prevent ATOH1 expression: when Notch is inhibited, ATOH1 is activated and drives cell cycle arrest, apoptosis, and terminal differentiation of progenitors into secretory cells. When both Notch and ATOH1 are inhibited, no change in proliferation or apoptosis is observed, and absorptive enterocyte differentiation proceeds normally. Therefore, Notch has no unique function in intestinal progenitors other than to regulate appropriate ATOH1 expression.
Our findings provide new insights into the mechanism of Notch signaling in the intestine. We found that Delta-like
Notch ligands are targets of ATOH1 activation in the intestine ( & , ). These ligands likely mediate lateral inhibition in the crypts (e.g., restriction of a subset of progenitors to the secretory fate), as shown in the zebrafish intestine 27
. Interestingly, we found that Jagged
ligands were not regulated by Notch-ATOH1 ( and unpublished observations), consistent with previous reports that they are targets of the WNT/β-catenin pathway 28
. Whether differential responses to distinct Notch ligands regulates the fate of differentiating progenitors or “quiescent” label-retaining stem cells versus “active” crypt-base columnar stem cells is an outstanding question for future investigation. We also found that reduction of ATOH1 induced HES1
expression in a Notch-dependent manner (). This indicates that ATOH1 and Notch reciprocally inhibit one another, suggesting a model of cell fate determination by lateral inhibition in which reciprocal inhibition and positive autoregulation combine to lock in secretory versus absorptive progenitor status.
Our results confirm that ATOH1 is critical for normal differentiation and homeostasis of intestinal progenitors 4, 26
. Recently, the cell cycle inhibitor p27Kip1
was identified as a target of Notch repression via Hes1, and was found to be de-repressed upon GSI treatment 9
. We found reduced p27 expression in Atoh1-mutant colons, which was not restored by Notch inhibition (Supplementary Figure 2
and Bossuyt et al 13
). Together, these data suggest a model for regulation of intestinal progenitor proliferation where p27 expression is activated by Atoh1 and repressed by Hes1. De-repression of p27 as a result of Notch/Hes1 inhibition allows increasing Atoh1 to enhance p27 expression and block proliferation. In Atoh1
-null cells, reduced p27 is associated with increased proliferation; de-repression of p27 by Notch/Hes1 inhibition is insufficient without coordinate activation of p27 by Atoh1, and thus proliferation is maintained.
Our findings support the hypothesis that ATOH1’s tumor suppressive function 12–13, 29
is mediated by its effects on intestinal progenitors and/or cancer stem cells. Consistent with this tumor suppressive function, we find hyperproliferation and reduced apoptosis of Atoh1-null
adenomas ( and supplemental Figure 3
). Of note, cancer cells respond to GSI not by inducing apoptosis but instead by reducing proliferation in an Atoh1-dependent manner (compare normal progenitors in and Supplemental Figure 3
to cancer cells in and and Supplemental Figure 3 and 4
). Atoh1-dependent, GSI-induced cell cycle arrest may enhance tumor cell killing by cytotoxic drugs 21–23
; whether ATOH1 mediates the synergistic effect of GSI and chemotherapeutics should be examined in the future.
Our results have several therapeutic implications. GSI therapy for non-intestinal malignancies and other diseases has been limited due to toxicity in the intestine 18, 30
. Our results suggest that targeted reduction of ATOH1 may prevent these side effects, and also that the expression level of ATOH1 is predictive of any approach to mitigating intestinal toxicity. As one approach to mitigate intestinal toxicity of GSIs, Real et al
. showed that glucocorticoid treatment blocked the effects of GSIs in the intestine while enhancing the anti-leukemic and lymphoid effects 30
. The ability of glucocorticoid to block intestinal toxicity of GSIs was suggested to be mediated by downregulation of the transcription factor Klf4; whether Atoh1 is involved in this process remains to be determined. Recently, Notch-sparing GSIs have been developed to selectively inhibit processing of Aβ in Alzheimer’s disease 16
. We suggest that ATOH1 expression may be a useful functional marker of intestinal side effects when determining the relative selectivity of the GSIs for Aβ over Notch. Among GI malignancies, we show that ATOH1 is a critical mediator of GSI effects. Therefore, the ability to induce ATOH1 is key for these therapies to be effective. Importantly, we recently showed that ATOH1 is silenced in ~80% of sporadic colorectal cancers 13
; therefore GSI therapy of these silenced tumors will be ineffective since they lack expression of the critical mediator of GSI treatment (ATOH1
). To utilize GSIs in GI malignancies, we suggest that patients should first be stratified according to ATOH1 silencing status. For example, we predict that ~20% of CRCs that retain ATOH1 expression will respond to GSI therapy. Furthermore, we predict that gastric and esophageal tumors will respond to GSI therapy when they include ATOH1-positive intestinal metaplasia, but will become resistant to GSI therapy if they progress further and silence ATOH1. Together, our results demonstrate that ATOH1 is essential for all effects of Notch/γ-secretase inhibitors in normal and cancerous intestinal cells, and suggest that selective targeting of ATOH1 will allow for tissue specific therapeutic modalities. More broadly, our results support the concept of targeting tissue-specific differentiation factors as an important approach to inhibiting growth of cancers.