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

Disclosures Nothing to Disclose

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

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

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

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

Aim Which MMP9 protects from CAC ?

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

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

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 *

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

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

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

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

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

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

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

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

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