Epithelial derived-matrix metalloproteinase (MMP9) exhibits a novel defensive role of tumor suppressor in colitis associated cancer by activating MMP9-Notch1-ARF-p53 axis

Abstract

Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9−/−, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9−/− mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway “MMP9-Notch1-ARF-p53 axis” regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.

Matrix metalloproteinase MMP9 maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer

“…All animal procedures were in compliance with the Guide for the Care of Use of Laboratory Animals from the US Public Health Service and with approval from the Animal Care Committee of Georgia State University. As described previously [ 15 ], 10 weeks old gender matched TgM9 and their wild-type (WT) littermates of C57/B6 background were used for the study. Comprehensive characterization of TgM9 have been done and published by our group [ 14 , 15 ].…”

“…As described previously [ 15 ], 10 weeks old gender matched TgM9 and their wild-type (WT) littermates of C57/B6 background were used for the study. Comprehensive characterization of TgM9 have been done and published by our group [ 14 , 15 ]. Mice were maintained on a 12-hour dark-light cycle and allowed free access to no purified diet pellets and tap water.…”

“…Animals were divided into two groups- one as control group, exposed to water only (without CAC) and another as experimental group with CAC induction. As described previously [ 15 ], CAC was induced among both TgM9 mice (n=30) and WT (n=30) mice by 3 cycles of dextran sodium sulfate (DSS) (MP Biomedicals, Salon, OH). Control group including both TgM9 and WT mice were exposed to drinking water only.…”

“…We and others have previously shown that MMP9 is a mediator of acute colitis [ 11 , 14 , 18 , 19 ] and colorectal cancer [ 20 – 22 ]. We have shown that MMP9 has a contrasting protective role in CAC in the setting of chronic inflammation and, acts as a tumor suppressor in CAC [ 12 , 13 , 15 , 23 ]. Considering the fact that GI tract is mainly constituted of epithelial cells and colon cancer has epithelial origin, we have generated transgenic mice ‘Tg-villin-MMP9 (TgM9)’ that constitutively expresses MMP9 under villin promoter (TgM9) [ 14 , 15 ].…”

“…We have shown that MMP9 has a contrasting protective role in CAC in the setting of chronic inflammation and, acts as a tumor suppressor in CAC [ 12 , 13 , 15 , 23 ]. Considering the fact that GI tract is mainly constituted of epithelial cells and colon cancer has epithelial origin, we have generated transgenic mice ‘Tg-villin-MMP9 (TgM9)’ that constitutively expresses MMP9 under villin promoter (TgM9) [ 14 , 15 ]. The aim of this study is to investigate the mechanism by which MMP9 maintains the epithelial-mucosal integrity in CAC, as the ‘extrinsic mechanistic pathway’.…”