Project description:Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer-related death worldwide Unique combinations of mutations can affect responsiveness to specific therapeutics. This can be, at least in part, due to mutation-specific alterations in gene regulatory mechanisms. To better understand how unique combinations of mutations affect gene regulation, we generated small RNA-seq, length extension chromatin run-on-seq (leChRO-seq), and RNA-seq data from APC (A-mutant), APC/KRAS (AK-mutant), APC/KRAS/TP53 mutant (AKP-mutant), and iGFP control human colonic organoids. From these analyses, we found that our colonic organoid models demonstrate mutation-specific patterns of miRNA transcription, miRNA expression and transcriptional regulatory element actvitiy. Furthermore, we defined 10 patterns of miRNA expression across organoid models. We highlighted one group of miRNAs that exhibited a unique downregulation of expression in AKP-mutant organoids (including miR-34a-5p and miR-10a-5p). Analysis of miRNA transcription revealed that most changes in miRNA expression are correlated with changes in miRNA transcription. Analysis of leChRO-seq data revealed that transcriptional regulatory elements upregulated in AKP-mutant colonic organoids have an enrichment of predicted binding sites for oncogenic transcription factors.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description: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.
Project description:We isolated and selected intestinal adenoma organoids from villin-CreER; Apcflox/flox and villin-CreER; Apcflox/flox; Prox1flox/flox mice and added tamoxifen to induce the deletion of the Apc and Prox1 genes in the intestinal epitheliul ex vivo. Microarray experiments were carried out 7 days after the addition of tamoxifen. Total RNA obtained from villin-CreER; Apcflox/flox and villin-CreER; Apcflox/flox; Prox1flox/flox organoids were compared 7 days after the addition of tamoxifen and 5 days after the selection for Apc-mutant organoids in the absence of the Wnt-agonist R-Spondin1.
