1. Overexpression of MMP9 in colonic epithelium mediates protection in colitis associated cancer
PALLAVI GARG
Ph.D.

2. Disclosures
Nothing to Disclose

3. Matrix Metalloproteinases (MMPs)
Are a family of zinc-dependent proteolytic enzymes with the ability to degrade extracellular matrix substrates such as collagen, gelatin and proteoglycans

4. Colitis Associated Cancer (CAC) & Colorectal Cancer
CAC is an important complication of Ulcerative Colitis or colonic Crohn’s Disease that results in significant morbidity and mortality
It causes 1/6 of all deaths in patients with ulcerative colitis
Colon cancer develops in flat dysplastic tissue among IBD individuals
CAC is often multiple, anaplastic, broadly infiltrating, rapidly growing and occurs at a younger age
The pathogenesis of CAC is not known

5. MMP9 and its role in acute colitis
MMP9 is not expressed in normal tissues and is one of the predominant MMPs that is up-regulated in several animal models of colitis and human IBD
MMP9 mRNA and protein levels are increased in the inflamed colonic mucosa of patients with IBD
MMP9 activity correlates with active inflammation
MMP9 activates Notch1 signaling in colonic epithelium
Castaneda et al, 2005, Gastroenterology
Garg et al, 2007, Gastroenterology

6. Role of MMP9 in CAC MMP9-/- mice show increased susceptibility to CAC
CAC in MMP9-/- mice is associated with decreased apoptosis compared to WT mice
CAC in MMP9-/- mice showed decreased levels of p21WAF1/Cip1 and p53
MMP9 mediates its tumor suppressive role via activation of Notch1 signaling
Garg et al, 2010, Cancer Research
Garg et al, 2011, Gastroenterology

7. Aim
Which MMP9 protects from CAC ?

8. In vivo model C57/B6 WT Tg-villin-MMP9 Water DSS (3 cycles) Water
DSS= Dextran Sodium Sulfate (3% in drinking water)

9. In vivo protocol Days 49 7 14 28 35 DSS Cycle (1st) starts
Days 49 7 14 28 35
DSS Cycle (1st) starts
DSS Cycle (2nd ) starts
Recovery
(1st) starts
(2nd) starts
DSS Cycle (3rd ) starts
(3rd) starts 53 85
sacrifice

10. Overexpression of epithelial MMP9 exhibited resistance to CAC*
Body weight gain in percentage
DSS cycle 1
Recovery cycle 1
DSS cycle 2
Recovery cycle 2
Sacrifice
DSS cycle 3
Recovery cycle 3 1 2 3
Tg-villin-MMP9 WT *

11. Overexpression of epithelial MMP9 exhibited less tumor incidence in CAC
Number of polyps
Number of dysplastic lesions *
WT CAC
Tg-villin-MMP9 CAC
WT CAC
Tg-villin-MMP9 CAC

12. Overexpression of epithelial MMP9 exhibited resistance to CAC
WT CAC
Tg-villin-MMP9 CAC

13. Overexpression of epithelial MMP9 showed increased levels of Notch1and p53 in CAC
Tg-villin-MMP9, CAC A
WT, CAC
MMP9, 104 kD
β-tubulin 1 2 3 4 5 6
Tg-villin-MMP9, CAC
WT, CAC B
NICD, 80 kD
β-tubulin 1 2 3 4 5 6
Tg-villin-MMP9, CAC
WT, CAC C
p53, 53 kD
GAPDH 1 2 3 4 5 6

14. Overexpression of epithelial MMP9 showed increased levels of p21WAF1/Cip1and Bax1 in CAC
Tg-villin-MMP9, CAC
WT, CAC A
p21WAF1/Cip1, 21kD
GAPDH 1 2 3 4 5 6
Tg-villin-MMP9, CAC B
WT, CAC
Bax-1, 37 kD
GAPDH 1 2 3 4 5 6

15. Overexpression of MMP9 in MMP9-/- MEFs showed increased levels of Notch1 and p53
WT MEFs
MMP9-/-MEFs + MMP9 A
MMP-9, 104 kD B
NICD, 80 kD
GAPDH 1 2 3 4 5 6
MMP9-/- MEFs
WT MEFs
MMP9-/-MEFs + MMP9 C
p53, 53 kD
GAPDH 1 2 3 4 5 6

16. Summary Epithelial-derived MMP9 plays a tumor suppressive role in CAC
Overexpression of MMP9 in colonic epithelium is associated with altered levels of p53, Notch1, Bax1 and p21WAF1/Cip1
Overexpression of MMP9 in MMP9-/-exhibited increased levels of NICD and p53

17. - Molecular mechanism of p53 regulation by MMP9 via Notch1
Inflammation
nucleus
ARF +
ARF
Mdm2 +
MMP-9 +
Notch-1 +
Mdm2 -
p53 +
cytosol

18. Conclusions
Despite being a mediator of acute inflammation, epithelial derived- MMP9 acts as a tumor suppressor in CAC
MMP9 mediated p53 activation via Notch1 signaling is necessary and sufficient for its tumor suppressive effect in CAC

19. Acknowledgement Didier Merlin
Lewins Walter
Crohn’s & Colitis Foundation of America Career Development Award, (#3057)