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Apc-mutants act as supercompetitors in intestinal tumour initiation

ABSTRACT: Almost all colorectal cancers (CRC) present with mutations in the Apc gene, leading to unrestrained Wnt activation and the initiation of tumour development. We previously reported the competitive benefit of Apc-mutant intestinal stem cells (ISCs) within the crypt, however, the mechanism by which they outcompete their wild type (WT) neighbours remained elusive. Here, we studied the effect of Apc-mutants using an in vitro culture system of WT (Lgr5-CreErt2) and Apc-/- (Lgr5-CreErt2;Apcfl/fl) organoids . The expression patterns of WT and Apc-/- organoids revealed significant upregulation in specific secreted Wnt antagonists Notum, Wif1 and Dkk2. Furthermore, we studied the effect of the secreted antagonist by treating WT organoids with either WT or Apc-/- conditioned medium (CM) for 48 hours and observed a significant decrease in stem cell markers and an increase in goblet cell markers. We report that Apc-mutants act as bona fide supercompetitors by secreting Wnt antagonists that result in active differentiation of WT ISCs.

ORGANISM(S): Mus musculus

PROVIDER: GSE144325 | GEO | 2021/03/25

REPOSITORIES: GEO

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Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

Project description:The adult stem cell marker Lgr5 and its close relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. Conditional deletion of the two genes in the gut impairs expression of Wnt target genes and results in rapid demise of intestinal crypts, thus phenocopying the effects of Wnt pathway inhibition. By mass-spectrometry, we find that Lgr4 and Lgr5 associate with the endogenous Wnt co-receptors Frizzled-5/7 and Lrp5/6 in HEK293 cells and in colorectal cancer cells. We also find that soluble Rspondin1, a Wnt pathway agonist, specifically binds to endogenous Lgr4 on HEK293 cells. In these cells, soluble Rspondin1 potently enhances canonical Wnt signals initiated by Wnt3A. Removal of Lgr4 does not affect Wnt3A-induced signals, but abrogates the Rspondin1-mediated enhancement of these signals, a phenomenon rescued by re-expression of Lgr4, -5 or -6. Rspondin1 and can be rescued by Wnt pathway activation. Lgr4/5/6 are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble Rspondin proteins. We used two different experimental setups to show that concomitant deletion of Lgr4 and Lgr5 affects the expression of Wnt pathway target genes. First, we determined the downregulated genes after Lgr4/5 deletion in AhCre_Lgr4fl/fl_Lgr5 fl/fl mice on day 1 post-deletion, before any histological changes are apparent in these mice (Lgr4/5 gene signature). Next, we acutely withheld the Wnt agonist Rspondin1 from in vitro crypt organoid cultures and performed differential gene expression before, and 1 day after withdrawal (Rspondin1 withdrawal signature). For better comparison we also deleted the Lgr4 and Lgr5 in vitro in organoids isolated from VillinCre_Lgr4fl/fl_Lgr5 fl/fl mice, and performed microarrays 1 day after deletion in vitro. Furthermore, we used published expression data from Apcfl/fl mice (Apc deletion signature). Deletion of Apc results in the immediate upregulation of Wnt pathway target genes. Comparison of the Lgr4/5 gene signature to both the Rspondin1 withdrawal signature and the Apc deletion signatures using Gene Set Enrichment Analysis (GSEA) showed that the genes deregulated after simultaneous deletion of Lgr4 and -5 are in the majority under the control of the Wnt pathway.

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Apc-mutants act as supercompetitors in intestinal tumour initiation

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