Post on 05-Feb-2016
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Chromatin remodelling ATPase Brg-1 is an essential factor for the maintenance of the intestinal crypt stem cell and adenoma
formation
Aliaksei Holik
BRG-1 and cancer
BRG-1 mutations are found in human cancer cell lines and in primary cancers
Heterozygous deletion of Brg-1 in mice promotes tumourigenesis via Brg-1 haploinsufficiency
Restoration of BRG-1 function leads to the reversion of a cancer phenotype
c-Fos
BRG-1
BRCA1 RB/p21
p53
Wnt-pathway
Suggested mechanisms of BRG1 role in tumourigenesis
Wnt pathway
Willert K., Jones K. A. Genes Dev. 2006;20:1394-1404
Small intestine architecture
Villus
Crypt
Stem cell
Transit amplifying cells
loxP strategyGeneration of conditional knock-out using Cre-loxP techniques
Cre recombinase Ah ER Cre recombinase Villin ER
BRG1 loxP loxP
BRG1 loxP loxP
Brg-1-deficient cells in small intestine are repopulated with wild-type cells
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
Ratio
of r
ecom
bine
d cr
ypts
Day 3 Day 5 Day 7BRG-1
AhCre-ERT
Brg-1-deficient small intestinal epithelium appears to be normal at day 4 post induction
Brg1fl/fl
Control
BRG-1
VillinCre-ERT
Brg1-deficient small intestinal epithelium displays no difference in mitosis and
apoptosis levels
Frequency of Mitosis and Apoptosis Per Crypt
0
0.2
0.4
0.6
0.8
1
1.2
Mitosis Apoptosis
VillinCre-Brg1fl/fl
VillinCre+Brg1fl/fl
p-values:
Mitosis – 0.629
Apoptosis – 0.057
Brg1-deficient small intestinal epithelium displays no difference in mitosis and
apoptosis levels
VillinCre-Brg1fl/fl
VillinCre+Brg1fl/fl
Apoptosis Position Distribution
0
0.5
1
1.5
2
2.5
3
3.5
1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 25-27 28-30 31-33
Cell Position
Inactivation of Brg-1 in small intestinal epithelium leads to rapid crypt loss
Brg1fl/fl
Control
Day 5 Day 7Day 4
Brg-1 loss leads to the failure of the crypt stem cell
Active stem cell
Brg1+ Brg1-Brg1-Transit amplifying cells
Relative Quantity of Ascl2 and Lgr5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Ascl2 Lgr5
Loss of Brg-1 results in reduced expression of intestinal stem cell markers
VillinCre-Brg1fl/fl
VillinCre+Brg1fl/fl
p-value < 0.05
Generation of double knock-out using Cre-loxP techniques
X BRG1
Cre recombinase Ah
loxP loxP
Mouse 1 Mouse 2
Cre recombinase Ah
BRG1 loxP loxP
Cre recombinase Ah
ER
ER
ER
APC loxP loxP
APC loxP loxP
Brg-1/Apc double mutant cells are selectively eliminated in small intestine
Small intestine of Brg1fl/fl/Apcfl/fl mouse at day 7 post induction
BRG-1-catenin
Brg-1-deficiency rescues lethal phenotype of Apc deletion in double knock-out mice
Control
Brg1fl/flApcfl/fl
Apcfl/fl
Brg1fl/fl
Median survival Apcfl/fl=8 Brg1fl/flApcfl/fl=14, P=0.0010
Brg-1 deletion removes a portion of Apc-deficient cells decreasing tumour
burden
Brg1-/Apc-
Brg1- Apc-
Brg-1 deletion removes a portion of Apc-deficient cells decreasing tumour
burden
Brg1-/Apc-
Brg1- Apc-
Microadenoma
Adenoma
Stem cell
Transit amplifying cells
Adenoma progression depends on the cell of origin
Conclusions
Brg-1 is a crucial factor for the maintenance of the adult tissue stem cell in small intestine.
Brg-1 loss is incompatible with activation of the Wnt pathway in the murine small intestine
Targeting the intestinal stem cell provides an attractive concept for the removal of potentially malignant lesions in individuals with genetic predisposition to colorectal cancer
Acknowledgements
The Darwin Trust of Edinburgh
Alan Clarke
Boris Shorning
ARC group