Transcript PosterA0
Chromatin remodelling ATPase Brg-1 is an essential factor for the maintenance of the intestinal crypt stem cell and adenoma formation Aliaksei Holik, Boris Shorning, Alan Clarke The Darwin Trust of Edinburgh Results Abstract Brg-1 is a crucial factor for small intestinal stem cell maintenance It is getting more apparent that epigenetic modifications are playing a crucial role in the cancer development. The aim of this project is to investigate the functional interaction between ATPase subunit of SWI/SNF chromatin remodeling complex Brg-1 and Wnt pathway. Using conditional knock-out mice we have deleted Brg-1 in murine small intestine both in a normal and a Wnt-activated background. Simultaneous deletion of Brg-1 and Apc leads to faster repopulation of small intestine by Brg-1 proficient cells with no Brg-1/Apc double mutant cells observed at any time. Apc deficiency in murine small intestine confers a severe phenotype of gut failure, which leads to prompt animal death. Additional deletion of Brg-1 rescues this phenotype increasing animal survival. Overall, our results suggest that Brg-1 is a crucial factor for intestinal stem cell maintenance in both normal and Wnt-activated background and may serve as a potential target for anti-cancer therapy in Wnt-activated tumours. Ratio of recombined crypts 0.6 0.5 0.4 0.3 0.2 0.1 0 3 5 7 10 --catenin Day 3 Day 5 BRG-1 knock-out leads to the development of mammary tumours and accelerated rate of induced lung carcinogenesis in heterozygous mice, whereas null mice die at the preimplantation stage BRG-1 has been shown to interact with -catenin and promote expression of Wnt target genes in vitro Aims Day 7 Low frequency inactivation of Brg-1 in small intestinal epithelium leads to repopulation of Brg-1 deficient cells Inactivation of Brg-1 in the context of activated Wnt pathway in small intestine leads to selective elimination of double mutant cells and increases animal survival Brg1- -BRG-1 Brg1fl/fl ApcBrg1-/Apc- Elimination of the Brg-1-deficient cells in small intestine reduces overall number of Apc-deficient cells thus reducing tumour burden and increasing animal survival BrdU Positive Cells Per Half Crypt Apoptosis per Crypt 12.00 3.00 10.00 2.50 8.00 2.00 Microadenoma 6.00 1.50 4.00 1.00 2.00 0.50 0.00 0.00 Control Brg Hom Control Brg Hom High frequency inactivation of Brg-1 in small intestinal epithelium does not affect cell proliferation and decreases apoptosis rate at day 4 after induction Adenoma Selective elimination of Brg-1 dependent stem cells in the context of activated Wnt signalling prevents development of advanced adenomas contributing to the animal survival (Barker et al., Nature, 2009) Control Conclusions Brg1fl/fl Day 4 Day 5 Day 7 Brg-1 To investigate the in vivo effects of Brg-1 deficiency alone and in the Wnt activated background using a conditional mouse knock-out model To search for novel therapeutic targets for cancers with activated Wnt-signalling -BRG-1 Control BRG-1 in Cancer e.g. RB, BRCA1, p53, c-Fos, c-Myc 14 Days Post Induction Background is an ATPase subunit of the SWI/SNF class of chromatin remodeling complexes It has been found mutated in a number of cancer cell lines and primary cancers It has been shown to interact with known tumour suppressors and oncogenes Brg-1 inactivation rescues otherwise lethal phenotype of Apc deficiency Timeline of gut repopulation Inactivation of Brg-1 in the small intestine leads to rapid repopulation of the epithelium with wild type cells. High-frequency deletion of Brg-1 results in extensive crypt death without increasing apoptosis levels of differentiated cells. These observations suggest that Brg-1 deficiency disrupts the ability of intestinal stem cells to maintain crypt function. BRG-1 This project is funded by High frequency inactivation of Brg-1 in small intestinal epithelium leads to rapid crypt loss is a crucial factor for intestinal stem cell maintenance in both normal and Wnt-activated background Targeting the active sub-population of intestinal stem sells might provide an attractive approach for the removal of potentially malignant lesions in patients with genetic predisposition for the colorectal cancer.