Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Lgr4/5/6 family members reside in Frizzled-Lrp complexes and mediate signaling by the Wnt agonist Rspondin1

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Daniel Stange

PROVIDER: E-GEOD-28265 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.

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Lgr4/5/6 family members reside in Frizzled-Lrp complexes and mediate signaling by the Wnt agonist Rspondin1

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