The APC tumour suppressor gene is the most commonly mutated gene in colorectal cancer (CRC). Loss of Apc in intestinal stem cells (ISCs) drives aberrant Wnt signalling and adenoma formation in mice 1 . We previously showed that a reduction in WNT-ligand secretion increases the ability of Apc-mutant ISCs to colonise a crypt (fixation) and accelerate tumourigenesis 2 . Here, we investigate key mechanistic processes whereby Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We find that Apc-mutant cells are enriched for transcripts encoding several secreted Wnt antagonists, with Notum being the most highly expressed. Indeed, conditioned medium from Apc-mutant cells suppresses the growth of wild-type organoids in a Notum-dependent manner. Furthermore, Notum-secreting mutant clones actively inhibit the proliferation of surrounding wild-type crypt cells and drive their differentiation, thereby outcompeting them from the niche. Importantly, genetic or pharmacological inhibition of Notum is sufficient to abrogate the expansion of Apcmutant cells and their ability to form intestinal adenomas. Taken together, we demonstrate Notum as a key mediator during the early stages of mutation fixation, which can be targeted to restore wild-type cell competition and thus, offer novel preventative strategies for high-risk patients. MainThe colonic epithelium displays one of the highest mutation rates of all tissues 3,4 , with lossof-function mutations in the APC tumour suppressor considered a key early event in colorectal cancer (CRC) initiation 5 . For a mutation to be maintained within a crypt, it needs to become "fixed", by mutant cells outcompeting wild-type intestinal stem cells (ISC) from the crypt 6,7 .Previous studies revealed that Apc loss (or Kras activation) confer a clonal advantage to ISCs 7,8, increasing their probability of fixation/winning within the crypt and, in the case of Apc mutation, driving adenoma formation. Even though APC-deficient clones have an increased probability of "winning", they can still be stochastically eliminated from the ISC pool i.e. lose.This suggests uncovering the molecular mechanisms by which APC-deficient cells outcompete wild-type cells could lead to novel chemo-preventative approaches.APC is a negative regulator of Wnt signalling that functions as an integral part of the destruction complex, which directs the phosphorylation and degradation of β-catenin 9 . Since Apc-mutant tumours exhibit constitutive Wnt-pathway activation, we first sought to identify genes differentially upregulated in Apc-mutant cells relative to the normal intestinal epithelium.For this, we performed transcriptomic analysis of tumours that develop in VillinCre ER ;Apc fl/+ (hereafter VilCre ER ;Apc fl/+ ) mice following the sporadic loss of the remaining copy of Apc 10 , akin to human CRC 11 . As expected, Wnt-target genes were highly upregulated in these Apcmutant tumours (Extended Data Fig. 1a). The most highly upregulated gene was Notum (Fig. 1a), which encodes a secreted WNT...
The homeobox transcription factor PROX1 is induced by high Wnt/β-catenin activity in intestinal adenomas and colorectal cancer, where it promotes tumor progression. Here we report that in LGR5 colorectal cancer cells, PROX1 suppresses the Notch pathway, which is essential for cell fate in intestinal stem cells. Pharmacologic inhibition of Notch in 3D organoid cultures from transgenic mouse intestinal adenoma models increased expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferation of the PROX1-positive colorectal cancer cells, but did not affect their ability to give rise to PROX1-negative secretory cells. Conversely, deletion increased Notch target gene expression and promoter activity, indicating reciprocal regulation between PROX1 and the Notch pathway in colorectal cancer. PROX1 interacted with the nucleosome remodeling and deacetylase (NuRD) complex to suppress the Notch pathway. Thus, our data suggests that PROX1 and Notch suppress each other and that PROX1-mediated suppression of Notch mediates its stem cell function in colorectal cancer. These findings address the role of the PROX1 homeobox factor as a downstream effector of Wnt/β-catenin singling in colorectal cancer stem cells and show that PROX1 inhibits the Notch pathway and helps to enforce the stem cell phenotype and inhibit differentiation. .
Transcription profiling data (single cell RNA sequencing) can be accessed in Gene Expression Omnibus with accession number GSE146139 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146139)
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