Erin E. Witalison

Protein Arginine Deiminases and Associated Citrullination: Physiological Functions and Diseases Associated with Dysregulation

Human proteins are subjected to more than 200 known post-translational modifications (PTMs) (e.g., phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, Nacetylation, and citrullination) and these PTMs can alter protein structure and function with consequent effects on the multitude of pathways necessary for maintaining the physiological homeostasis. When dysregulated, however, the enzymes that catalyze these PTMs can impact the genesis of countless diseases. In this review, we will focus on protein citrullination, a PTM catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Specifically, we will describe the roles of the PADs in both normal human physiology and disease. The development of PAD inhibitors and their efficacy in a variety of autoimmune disorders and cancer will also be discussed.

Molecular targeting of protein arginine deiminases to suppress colitis and prevent colon cancer

Ulcerative colitis (UC) is a chronic disease, in which the lining of the colon becomes inflamed and develops ulcers leading to abdominal pain, diarrhea, and rectal bleeding. The extent of these symptoms depends on disease severity. The protein arginine deiminase (PAD) family of enzymes converts peptidyl-Arginine to peptidyl-Citrulline through citrullination. PADs are dysregulated, with abnormal citrullination in many diseases, including UC and colorectal cancer (CRC). We have developed the small molecule, pan-PAD inhibitor, Chlor-amidine (Cl-amidine), with multiple goals, including treating UC and preventing CRC. Building off our recent results showing that: 1) Cl-amidine suppresses colitis in vivo in a dextran sulfate sodium (DSS) mouse model; and 2) Cl-amidine induces microRNA (miR)-16 in vitro causing cell cycle arrest, we tested the hypothesis that Cl-amidine can prevent tumorigenesis and that miR-16 induction, by Cl-amidine, may be involved in vivo. Consistent with our hypothesis, we present evidence that Cl-amidine, delivered in the drinking water, prevents colon tumorigenesis in our mouse model of colitis-associated CRC where mice are given carcinogenic azoxymethane (AOM), followed by multiple cycles of 2% DSS to induce colitis. To begin identifying mechanisms, we examined the effects of Cl-amidine on miR-16. Results show miR-16 suppression during the colitis-to-cancer sequence in colon epithelial cells, which was rescued by drinking Cl-amidine. Likewise, Ki67 and cellular proliferation targets of miR-16 (Cyclins D1 and E1) were suppressed by Cl-amidine. The decrease in cell proliferation markers and increase in tumor suppressor miRNA expression potentially define a mechanism of how Cl-amidine is suppressing tumorigenesis in vivo.

Inhibiting Protein Arginine Deiminases Has Antioxidant Consequences

Ulcerative colitis is a dynamic, idiopathic, chronic inflammatory condition that carries a high colon cancer risk. We previously showed that Cl-amidine, a small-molecule inhibitor of the protein arginine deiminases, suppresses colitis in mice. Because colitis is defined as inflammation of the colon associated with infiltration of white blood cells that release free radicals and citrullination is an inflammation-dependent process, we asked whether Cl-amidine has antioxidant properties. Here we show that colitis induced with azoxymethane via intraperitoneal injection + 2% dextran sulfate sodium in the drinking water is suppressed by Cl-amidine (also given in the drinking water). Inducible nitric oxide synthase, an inflammatory marker, was also downregulated in macrophages by Cl-amidine. Because epithelial cell DNA damage associated with colitis is at least in part a result of an oxidative burst from overactive leukocytes, we tested the hypothesis that Cl-amidine can inhibit leukocyte activation, as well as subsequent target epithelial cell DNA damage in vitro and in vivo. Results are consistent with this hypothesis, and because DNA damage is a procancerous mechanism, our data predict that Cl-amidine will not only suppress colitis, but we hypothesize that it may prevent colon cancer associated with colitis.

Looking for the best anti-colitis medicine: A comparative analysis of current and prospective compounds

Ulcerative colitis (UC) is a chronic lifelong inflammatory disorder of the colon, which, while untreated, has a relapsing and remitting course with increasing risk of progression toward colorectal cancer. Current medical treatment strategies of UC mostly focus on inhibition of the signs and symptoms of UC to induce remission and prevent relapse of disease activity, minimizing the impact on quality of life, but not affecting the cause of disease. To date, however, there is no single reliable treatment agent and/or strategy capable of effectively controlling colitis progression throughout the patients life without side effects, remission, or resistance. Taking into consideration an urgent need for the new colitis treatment strategies, targets and/or modulators of inflammation, we have tested current and prospective compounds for colitis treatment and directly compared their anti-colitis potency using a dextran sulfate sodium (DSS) mouse model of colitis. We have introduced a composite score – a multi-parameters comparison tool – to assess biological potency of different compounds.

Repurposing the anti-malarial drug, quinacrine: new anti-colitis properties

Background Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is associated with an increased risk of colorectal cancer in 8-10 years after disease onset. Current colitis treatment strategies do not offer a cure for the disease, but only treat the symptoms with limited success and dangerous side-effects. Also, there is no preventive treatment for either UC or colorectal cancer. Quinacrine is an anti-malarial drug with versatile use in the treatment of diseases involving inflammatory response such as rheumatoid arthritis and lupus erythematosus. It also has putative anti-cancer effect. Quinacrines anti-inflammatory, anti-oxidant properties, and anti-tumorigenic properties make it a potential small molecule preventive agent for both UC and associated colorectal cancer. Results There were obvious changes in the CDI, histology, and inflammatory load in quinacrine-treated groups in a dose and time dependent manner in both models of UC, induced by chemical or haptenating agent. Methods We tested quinacrine at two different doses as a colitis treatment agent in two mouse models of UC - the dextran sulfate sodium and oxazolone. The clinical disease index (CDI), histological changes of the colon, levels of inflammatory markers (Cox-2, iNOS, p53) and overall health vitals were evaluated. Conclusions We demonstrate that quinacrine successfully suppresses colitis without any indication of toxicity or side-effects in two mouse models of UC.

Citrullination in Inflammatory-Driven Carcinogenesis of the Colon

Chronic inflammatory diseases are often accompanied by an increased risk of carcinogenesis. Many stimuli can be attributed to the onset of chronic inflammation. In ulcerative colitis, specifically, protein arginine deiminase (PAD) expression and levels of citrullinated proteins are elevated and credited to the initiation of the disease. Subsequently, PAD levels are higher in human samples with inflammatory-driven carcinogenesis of the colon as compared to normal healthy tissue. PAD inhibition with Cl-amidine has successfully suppressed signs of inflammation and tumors in ulcerative colitis and colitis-driven carcinogenesis, respectively. The efficacy of Cl-amidine as a pan-PAD inhibitor indicates the essential role of PADs in ulcerative colitis and inflammatory-driven carcinogenesis of the colon.

Abstract 5503: Panaxynol, a potential treatment for colitis, selectively targets pro-inflammatory macrophages for DNA damage and apoptosis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) that has been associated with a high risk for colorectal cancer. Since available conventional treatments provide mostly modest relief and are paired with a high risk of side effects, complementary and alternative medicines (CAMs) can offer a safe and effective option for the treatment of IBD. Earlier studies in our lab have shown that American Ginseng can suppress colitis and also prevent colitis-associated colon cancer in mice. During the process of isolating active ingredients from this plant, Panaxynol was identified as a key component. Panaxynol has been shown to have anti-cancer and anti-inflammatory properties, but it has not been studied in macrophage-driven autoimmunity models. Here, we study the effects of Panaxynol on macrophages and other cell lines in an attempt to understand its mechanism of action. We show that Panaxynol induces DNA damage and apoptosis in macrophages in a dose-dependent manner using phosphorylated γ-H2AX detection and TUNEL assays, respectively. Importantly, DNA damage and apoptosis were induced in other cell types (e.g. epithelial cells and fibroblasts) only when using higher doses of Panaxynol (>50μM). Co-culture experiments confirm that Panaxynol selectively targets macrophages in the presence of colon cancer cells. To explore mechanisms, we asked whether Panaxynol inhibits the STAT1 pathway. Results showing an inhibition of STAT1 phosphorylation are consistent with the hypothesis. We plan to extend these studies to an in vivo mouse model of DSS-induced colitis in order to understand the anti-inflammatory mechanisms of Panaxynol. Positive outcomes from this study can potentially serve as a model for the treatment of colitis and other autoimmune diseases (at least partly) associated with macrophage dysfunction. Citation Format: Anusha Chaparala, Deepak Poudyal, Xiangli Cui, Bin Li, Erin E. Witalison, Taixing Cui, Lorne J. Hofseth. Panaxynol, a potential treatment for colitis, selectively targets pro-inflammatory macrophages for DNA damage and apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5503. doi:10.1158/1538-7445.AM2015-5503

Abstract 3782: Cl-amidine, a PAD inhibitor, prevents UC and CRC in mice: Exploring novel mechanisms of miRNA and oxidative stress regulation

Ulcerative Colitis (UC) is a chronic, relapsing inflammatory bowel disease that affects millions of patients worldwide. Due to the chronic inflammatory state of UC, which causes oxidative stress and leads to DNA damage, patients with UC are at a higher risk of developing colorectal cancer (CRC). Although the cause of UC is still unknown, the protein arginine deiminase (PAD) family of enzymes has been found to be a key component in many human inflammatory diseases and cancer, including UC and CRC. PADs are calcium dependent enzymes that post-translationally convert peptidyl-Arginine to peptidyl-Citrulline through ‘citrullination’. In hopes of treating UC and preventing CRC, we developed Chlor-amidine (Cl-amidine), a novel small molecule inhibitor of PADs. Cl-amidine irreversibly inhibits PADs by covalent modification of Cys645 at the active site of the PAD enzyme. Unlike current approved drugs for the treatment of UC, Cl-amidine shows no toxicity both in vitro and in vivo. Preliminary in vitro studies showed that Cl-amidine is able to suppress oxidative stress, DNA damage, and the inflammatory marker, iNOS. Correspondingly, we found similar results in our AOM/DSS mouse model of UC, as Cl-amidine suppressed colonic inflammation. In this UC model, Cl-amidine treatment also decreased reactive oxygen species (ROS) production by inflammatory cells and DNA damage in epithelial cells. We proceeded to look at the capability of Cl-amidine to prevent carcinogenesis in our AOM/DSS mouse model of UC-associated CRC and report that Cl-amidine treatment significantly decreased tumor incidence in this model. To further understand how Cl-amidine prevents carcinogenesis, we are currently exploring the effect of Cl-amidine on two separate mechanisms: 1) miRNA and 2) oxidative stress regulation. First, our recent studies have focused specifically on miR-16, a putative tumor suppressor, which is downregulated in cancers. Interestingly, we have found that Cl-amidine upregulates miR-16 expression in our in vitro and in vivo models of UC. We aim to determine if Cl-amidine acts through miR-16 to prevent UC and UC-associated CRC by suppressing DNA damage and/or by suppressing cell proliferation. The second mechanism that we are exploring involves the regulation of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. In vitro results reveal that Cl-amidine increased protein levels of these enzymes. We plan to measure ROS levels in Cl-amidine treated cells that are introduced to SOD, GPx, and catalase inhibitors. This will provide evidence that Cl-amidine is working through these specific enzymes to reduce ROS activity. Based on the efficacy of Cl-amidine in our models of UC and CRC, and its potential multi-targeted mechanism of action; our research has revealed that Cl-amidine is a cutting-edge, prospective drug therapy for UC patients. Citation Format: Erin E. Witalison, Xiangli Cui, Paul R. Thompson, Lorne J. Hofseth. Cl-amidine, a PAD inhibitor, prevents UC and CRC in mice: Exploring novel mechanisms of miRNA and oxidative stress regulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2014-3782