Project description:RNA sequencing of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in full medium (high Wnt/Rspo (20%)) or medium without Wnt3a-low Rspo (0.2%).
Project description:Transcriptomic profiles of 6 commercially-available human patient-derived gastrointestinal organoid lines were obtained and compared to transcriptomic profile of a commercially available human iPSC-induced colon organoid line. Transcriptomic profile of iPSC-derived human colon organoid line was compared after culture in either Corning growth-factor-reduced Matrigel (Corning 356231) or MilliporeSigma growth-factor-reduced ECMGel (E6909)
Project description:Wnt/b-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce b-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with an ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves casein kinase-1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing b-catenin turnover and propelling ligandindependent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.
Project description:It has been demonstrated that Ring finger protein 43 (RNF43) is overexpressed in colorectal cancer and mediates cancer cell proliferation. We found that RNF43 was frequently overexpressed in HCC, and knockdown of RNF43 could induce apoptosis and inhibit proliferation, invasion, colony formation and xenograft growth of HCC cells. Suggesting that RNF43 is involved in tumorigenesis and progression of HCC. We used microarrays to profile gene expression patterns before and after RNF43 knockdown, and identified differentially expressed genes during this process. HepG2 cells were transfected with RNF43 siRNA or negative control siRNA in triplicate. Forty-eight hours after transfection, total RNA was extracted,labeled and hybridized to HG-U133 Plus 2.0 arrays.
Project description:To identify the tumor heterogeneiety among a single organoid that recapitulate the human colon cancer tissues, we put a single organoid derived from single colon cancer stem cells into single-cell RNA-sequencing. Isolated single cells were processed with Fluidigm C1 Single Cell Auto Prep system. 71 cells and one bulk control were sequenced using HiSeq2500.
Project description:It has been demonstrated that Ring finger protein 43 (RNF43) is overexpressed in colorectal cancer and mediates cancer cell proliferation. We found that RNF43 was frequently overexpressed in HCC, and knockdown of RNF43 could induce apoptosis and inhibit proliferation, invasion, colony formation and xenograft growth of HCC cells. Suggesting that RNF43 is involved in tumorigenesis and progression of HCC. We used microarrays to profile gene expression patterns before and after RNF43 knockdown, and identified differentially expressed genes during this process.
Project description:The homologous E3 ubiquitin ligases RNF43/ZNRF3 negatively regulate WNT signalling activation. Recently, both genes have been found mutated in several types of cancers. Specifically, loss-of-function mutations result in adenoma formation in mouse small intestine. However, their role in liver pathology and cancer has not been explored yet. Here, we describe that hepatocyte-specific deletion of Rnf43/Znrf3 results in altered lipid metabolism and steatohepatitis, in the absence of exogenous dietary fat supplementation, with a remarkable increase in unsaturated lipids. Upon liver injury, Rnf43/Znrf3 deletion results in impaired hepatocyte regeneration and liver cancer, subsequent to an imbalance between differentiation and proliferation. Using three different liver organoid models (hepatocyte-, hepatoblast- and ductal cell-derived organoids) we demonstrate that the hepatocyte differentiation defects and the lipid metabolic alterations are, at least in part, cell-autonomous. Remarkably, hepatocellular carcinoma patients with mutations in either or both genes or in other WNT pathway components also present altered hepatic lipid metabolism and non-alcoholic steatohepatitis (NASH) profiles. Our findings imply that hyperactivation of WNT signaling through the RNF43/ZNRF3 module predisposes to liver cancer by impairing hepatocyte differentiation and altering the lipid metabolic ground-state of the liver. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 or WNT mutated individuals at risk of developing fatty liver and/or liver cancer.
Project description:In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal (CRC) patients. For most, organoids were also generated from adjacent normal tissue. The organoids closely resemble the original tumor. The spectrum of genetic changes observed 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 robotized, 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. We generated organoids from healthy tissue and coloncarcinoma tissue. The organoids were trypsinized, plated in matrigel and overlaid with medium. After three days, RNA was isolated using Qiagen RNAeasy. Medium conditions are the same for all organoids, irrespective of their origin.
Project description:To test the hypothesis that HNF4A should engage small intestine enhancers in colon and activate transcription, we over-expressed HNF4A in 2 independent mouse colonic orgnaoid lines and 5 independent human colonic organoid lines and performed RNA-seq studies. Both RNA-seq data revealed that small intestine functional pathways including fat, protein and carbohydrate digestion and absorption were activated after HNF4A OE. This gain-of-function study of HNF4A in mouse and human colon organoid supports HNF4A as a central mediator of small intestine gene activation in colonic plasticity, conserved across species.
Project description:RNF43-mutant pancreatic tumors are driven by Wnt signaling and sensitive to Wnt inhibition, e.g. PORCN inhibitors. However, some RNF43-mutant pancreatic cancers are intrinsically resistant to Wnt inhibition. We identified that EP300 mutations confer resistance to PORCN inhibitors in RNF43-mutant pancreatic cancers. We found that EP300 knockout down-regulated GATA6 expression and GATA6-regulated differentiation program, making the anti-differentiation roles of Wnt signaling dispensable.