BRG-1 in CancerBRG-1 is an ATPase subunit of the SWI/SNF class of chromatin remodeling complexesIt has been found mutated in a number of cancer cell lines and primary cancersIt has been shown to interact with known tumour suppressors and oncogenes e.g. RB, BRCA1, p53, c-Fos, c-Myc

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 stageBRG-1 has been shown to interact with -catenin and promote expression of Wnt target genes in vitro

Chromatin remodelling ATPase Brg-1 is an essential factor for the maintenance of the intestinal crypt stem cell and

adenoma formationAliaksei Holik, Boris Shorning, Alan Clarke

Abstract

Background

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.

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.

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.

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 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.

Results

This project is funded by The Darwin Trust of Edinburgh

Aims

CBP

Brg1 TCF

Groucho -catenin

-catenin

TCF

Wnt

-catenin

LRP

Fz

Dsh CK 1

Axin APC GSK -3

-catenin -catenin

Brg-1 is a crucial factor for small intestinal stem cell

maintenance

Low frequency inactivation of Brg-1 in small intestinal epithelium leads to repopulation of Brg-1 deficient cells

High frequency inactivation of Brg-1 in small intestinal epithelium leads to rapid crypt loss

Conclusions

Timeline of gut repopulation

0

0.1

0.2

0.3

0.4

0.5

0.6

3 5 7 10 14

Days Post Induction

Rat

io o

f re

com

bin

ed c

ryp

ts

Day 3 Day 5 Day 7

Microadenoma

Adenoma

Stem cel

l

Transit amplifying cells

Brg1-/Apc-

Brg1- Apc-

Day 4 Day 5 Day 7Brg1fl/fl

Control

BrdU Positive Cells Per Half Crypt

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Control Brg Hom

Apoptosis per Crypt

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Control Brg Hom

Brg-1 inactivation rescues otherwise lethal phenotype of Apc

deficiency

-BRG-1Brg1fl/fl

Control

High frequency inactivation of Brg-1 in small intestinal epithelium does not affect cell proliferation and decreases apoptosis rate at day 4 after induction

--catenin -BRG-1Inactivation 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

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

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)