Adani Pujada

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

In colitis associated cancer (CAC), chronic inflammation exposes the epithelial mucosal defensive lining to inflammatory mediators such as cytokines and anti-microbial peptides (AMPs) causing the dysbiosis of microbiota population and the dysregulation of immune response. Matrix Metalloproteinases (MMPs) are zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and carcinogenesis. MMP9 is undetectable in healthy tissue, although highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC opposite to its conventional role of acute inflammation and cancer mediator. In this study, we investigated the mechanistic role of MMP9 in preserving the epithelial mucosal integrity to suppress the progression of tumor microenvironment in CAC. We used transgenic mice constitutively expressing MMP9 in colonic epithelium (TgM9) as an in vivo model and intestinal cell line CaCo2BBE as an in vitro model. We induced CAC with three cycles of dextran sodium sulfate (DSS). We observed that MMP9 expression in colonic epithelium maintains the microbiota. We also observed that MMP9 mediates pro-inflammatory cytokine levels and AMPs but suppresses IL-22 resulting in lower levels of REG3-g and S100A8 AMPs. We also found that MMP9 maintains an efficient barrier function and the integrity of tight junctions. We also observed increased levels of mucin and intestinal trefoil factor among TgM9 mice in CAC. We also found that MMP9 expressing CaCo2BBE cells had increased expressions of EGFR and nuclear transcription factor- specificity protein 1 (Sp1). These data imply that MMP9 acts as a tumor suppressor in CAC by sustaining the epithelial mucosal integrity due to the activation of EGFR-Sp1 signaling pathway.

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

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.

Function, Regulation, and Pathophysiological Relevance of the POT Superfamily, Specifically PepT1 in Inflammatory Bowel Disease

Mammalian members of the proton-coupled oligopeptide transporter family are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs and couple substrate translocation to the movement of H+ , with the transmembrane electrochemical proton gradient providing the driving force. Peptide transporters are responsible for the (re)absorption of dietary and/or bacterial di- and tripeptides in the intestine and kidney and maintaining homeostasis of neuropeptides in the brain. These proteins additionally contribute to absorption of a number of pharmacologically important compounds. In this overview article, we have provided updated information on the structure, function, expression, localization, and activities of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4), and PhT2 (SLC15A3). Peptide transporters, in particular, PepT1 are discussed as drug-delivery systems in addition to their implications in health and disease. Particular emphasis has been placed on the involvement of PepT1 in the physiopathology of the gastrointestinal tract, specifically, its role in inflammatory bowel diseases.

Abstract 3670: Matrix metalloproteinase 9 has a novel tumor suppressive role in inflammation associated colorectal cancer by modulating intrinsic tumor microenvironment

Colitis associated cancer (CAC) is a deadly complication of inflammatory bowel disease, and a subtype of colon cancer. CAC malignancy progresses through enhanced DNA damage. Observance of microsatellite instability and chromosomal instability in non-cancer related inflammatory conditions, supports the fact that inflammation could contribute to genomic destabilization by generating reactive oxygen nitrogen species, producing double strand breaks leading to DNA damage. Thus chronic colitis can induce DNA damage and modulate DNA repair systems- components of the intrinsic pathway to modulate tumor microenvironment. Matrix metalloproteinases (MMPs) are the most prominent family of proteinases associated with almost all the junctures of inflammation and tumorigenesis. They mediate inflammation, tissue remodeling, invasion, metastasis and tumor growth. Among the 24 known human MMPs, MMP9 is very unique as it is undetectable in normal tissues but highly expressed in inflamed or ulcerated tissues. We have observed that MMP9 has a protective role in CAC, which is exclusive and contrary to its traditional role of a facilitator in acute inflammation and sporadic colon cancer. In this study we explore the molecular mechanism by which MMP9 prevents DNA damage by activating tumor suppressors and DNA repair pathways in CAC. We generated transgenic mice overexpressing MMP9 in intestinal epithelium (TgM9) and to mimic human CAC, 3 cycles of 3% DSS (dextran sodium sulfate, a colonic inflammation inducer) for 5 days, followed by two weeks of recovery was used for in vivo model. Stably-transfected HCT116 cells with/without p-EGFP-MMP9 plasmid were used for in vitro experiments. We have observed that in CAC, TgM9 mice had significantly decreased tumor incidence and dysplastic lesions and a lower histological score compared to their WT littermates. TgM9 mice exhibited increased apoptosis, protein expressions of active-Notch1, p19ARF, p53, p21WAF1/Cip1, caspase-3 and cyclinE in CAC compared to WTs. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation. FACS analysis indicated S-phase cell-cycle arrest and WB analysis of phosphorylated gamma-H2AX expressions indicated less DSBs compared to vector. HCT116 cells overexpressing MMP9 indicated increased expression of MDC1 and mis-match repair proteins MLH1. Our study highlights two novel findings i) MMP9 being a secretory protein activates transcellular protein Notch1 which translocates to nucleus and activates wildtype p53 via ARF and ii) MMP9 suppresses DNA double strand breaks by activating MMR gene- MLH1 and removing the damaged cells by apoptosis and or cell cycle arrest. Our study highlights the paradox of using MMP9 inhibitors in current therapies to treat CAC patients, implying that MMP9 expression might be a natural/ biological way to suppress inflammation associated ulceration. Citation Format: Lewins Walter, Adani Pujada, Sayeed Mohammadian, Agnieszka Bialkowska, Vincent Yang, Pallavi Garg. Matrix metalloproteinase 9 has a novel tumor suppressive role in inflammation associated colorectal cancer by modulating intrinsic tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3670.

Abstract 2675: MMP9 modulates ROS levels and DNA repair pathway to maintain genomic stability in colitis associated cancer

Introduction: Chronic inflammation predisposes tissues to oncogenic, and in the colon this condition is termed as colitis associated cancer (CAC). CAC is the inflammation-dysplasia-carcinoma pathway which is significantly different compared to the adenoma-carcinoma pathway of sporadic colon cancer (CRC). In CAC, chronic inflammation causes accumulation of reactive oxygen species (ROS) resulting in DNA damage that nurtures tumor microenvironment and accelerates cancer cell growth. Gut microbiota is important in regulating ROS levels, and DNA repair pathways are critical in restoring the genomic instability. Matrix Metalloproteinases (MMPs) are a family of zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and tumorigenesis. MMP9 is the most unique MMP because it is undetectable in healthy tissue but highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC which is opposite to its conventional role of mediator in acute inflammation and cancer. Aim: In this study, we examined that if MMP9 acts as a tumor suppressor by modulating gut microbiota and mismatch repair (MMR) genes to reinstate genomic stability in CAC. Methods: We used C57/B6 transgenic MMP9 mice which can express MMP9 under villin promoter (TgM9) and their wild type (WT) littermates. CAC was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS, inflammation inducer) to assess the microbiota population. As a proof of principal model, we used MMP9 small interfering RNA (siRNA) encoded within nanoparticles and gavaged WT mice to inhibit MMP9 in colonic epithelium. Results: QPCR data showed that mRNA levels of total microbiota population were more among TgM9 mice compared to WTs in CAC. QPCR analysis also indicated increased mRNA levels for the phylum Bacteroidetes sp., Akkermansia muciniphila but decreased mRNA levels for the phylum Firmicutes sp. TgM9 mice had lower levels of ROS compared to WTs in CAC. WB data showed decrease in protein expressions of γH2AX and MDC1 while an increase in protein expressions of MLH1 and pCNA compared to WTs in CAC. WT mice treated with MMP9 siRNA loaded nanoparticles showed worsened CAC condition compared to WT mice given scrambled siRNA loaded nanoparticles as reflected by significant bodyweight loss and higher clinical score. We also observed that WT mice treated with MMP9 siRNA loaded nanoparticles showed increase in protein expression of γH2AX while decrease in MLH1 protein levels compared to control group. Conclusion: Our study has two clinically relevant outcomes that MMP9 expression in CAC: i) promotes the beneficial microbiota population and controls the ROS production, ii) restores the DNA damage via activation of the MMR pathway. This implies the contradiction of the use of targeted MMP9 inhibitors in CAC treatment therapies by showing that MMP9 expression may be a natural way to suppress CAC. Citation Format: Lewins Walter, Adani Pujada, Brandon Canup, Hamed Laroui, Pallavi Garg. MMP9 modulates ROS levels and DNA repair pathway to maintain genomic stability in colitis associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2675. doi:10.1158/1538-7445.AM2017-2675

Matrix metalloproteinases as potential fecal biomarkers for ulcerative colitis – a function beyond their proteolytic activity

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