Published OnlineFirst March 20, 2019; DOI: 10.1158/0008-5472.CAN-18-2797 Cancer Review Research Citrullination in Cancer Arseniy E. Yuzhalin Abstract Posttranslational modifications of proteins have been impli- lination on different aspects of tumor biology, including (i) cated in pathogenesis of numerous diseases. Arginine deimina- regulation of apoptosis and differentiation, (ii) promoting EMT tion (also known as citrullination) has a principal role in and metastasis, and (iii) potential use of citrullinated antigens progression of rheumatoid arthritis through generation of for immunotherapy. In addition, (iv) the role of citrullination autoantibodies and exacerbation of the inflammatory response. as a cancer biomarker and (v) implication of neutrophil extra- Recently, multiple research groups provided solid evidence of cellular traps in tumorigenesis are discussed. In summary, citrullination being in control of cancer progression; however, current findings testify to the significance of arginine deimina- there is no comprehensive overview of these findings. This tion in tumor biology and thus more basic and translational article summarizes and critically reviews the influence of citrul- studies are needed to further explore this topic. Introduction and to a lesser extent periodontitis (12), autoimmune encepha- Citrulline is a noncoding amino acid produced in the body lomyelitis (13), and systemic lupus erythematosus (SLE; through a posttranslational deimination of peptidyl-arginine Fig. 1C; ref. 14). Here, deiminated proteins act as neoantigens (Fig. 1A; ref. 1). This reaction is catalyzed by peptidyl-arginine and inflict the production of autoantibodies, thus boosting a local deiminase (PAD) enzymes, which hydrolyze a guanidino group of inflammatory response and exacerbating the severity of dis- the arginine into a urea group, resulting in 1 Da change in ease (1). Importantly, deiminated proteins per se do not seem to molecular mass and converting a positively charged arginine into serve as an initial inflammatory trigger but uphold and intensify the electrically neutral citrulline, thereby affecting hydrogen bond an established inflammatory cycle; hence, citrullination cannot formation and protein folding, ultimately resulting in altered lead to the onset of a disease yet being able to stimulate its hydrophobicity, protein–protein interactions, or even causing progression and aggravation. Antibody responses to citrullinated denaturation (Fig. 1B; ref. 1–3). Human citrullinome is limited peptides are used to diagnose RA in clinical setting, whereas to several hundred proteins, most commonly including vimentin, multiple studies demonstrated that inhibition of PADs or asso- actin, filaggrin, collagen, fibronectin, keratin, tubulin, and various ciated citrullination alleviate RA in animal models (1). histones (4, 5). Besides its role in autoimmunity, evidence from multiple Exact biological function of citrullination remains obscured; translational and clinical studies pointed at the involvement of however, arginine deimination is recognized to be central for citrullination in multiple sclerosis, atherosclerosis, thrombosis, transcriptional regulation of gene expression (6). Citrullinated and inflammatory bowel disease (Fig. 1C). In these conditions, histones account for approximately 10% of all histone molecules, citrullination acts through quite different mechanisms. In neu- emphasizing the significance of this posttranslational modifica- rological disease, deimination of myelin basic protein reduces its tion in many nucleus-associated processes (7). Another known interactions with phospholipids, thereby inhibiting adherence function of citrullination is triggering the formation of neutrophil between layers of the myelin sheath and subsequently leading to extracellular traps (NET), a machinery to disable and eliminate demyelination (15–17). Pathogenesis of atherosclerosis and bacterial pathogens (8, 9). In neutrophils, PAD4-catalyzed citrul- thrombosis is aggravated due to NET formation by plaque- or lination of histones serves as a starting point for chromatin clot-infiltrating neutrophils, providing a feed-forward cycle for decondensation and subsequent NET release, enabling to combat disease progression (18). Along similar lines, NETs were detected infections (Fig. 1B; ref. 10). in crypt abscesses in colonic lesions of patients with ulcerative Arginine deimination is essential for pathogenesis of several colitis (19) whereas pharmacologic inhibition of PAD suppressed autoimmune diseases, mainly rheumatoid arthritis (RA; ref. 11), colitis in a mouse model of this disease (20). Despite inflammation is one of the hallmarks of cancer (21), there is a dearth of literature on the influence of citrullination on CRUK/MRC Oxford Institute for Radiation Oncology, Oxford, United Kingdom. tumor biology and progression. In this article, I summarize and Current addresses for A.E. Yuzhalin: Department of Molecular and Cellular critically review published studies on this topic. Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030; Research Institute for Complex Issues of Cardiovas- cular Diseases, 6 Sosnovy Blvd., Kemerovo 650002, Russia. Corresponding Author: Arseniy E. Yuzhalin, University of Oxford, ORCRB, Brief Overview of PAD Biology and Roosevelt Drive, Oxford OX3 7DQ, UK. Phone: 07983123963; Fax: 07983123963; Function in Physiologic Conditions E-mail: [email protected] þ PADs are a group of five Ca2 -dependent enzymes (PAD1–4 doi: 10.1158/0008-5472.CAN-18-2797 and PAD6), sharing 70% to 95% sequence homology and Ó2019 American Association for Cancer Research. expressed in a wide range of tissues and organs (Fig. 1D; www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 20, 2019; DOI: 10.1158/0008-5472.CAN-18-2797 Yuzhalin A B Guanidino group Uriedo group Consequences of arginine-to-citrulline conversion: H2N NH H2N O 1) Altered charge (positive to neutral); 2) Altered protein conformation and function; NH PADs NH 3) Changed cell-cell interactions; Ca2+ 4) Protein becomes susceptible to degradation. H H N N Biological processes involving citrullination: N H O NH N H O 2 3 H O 1) NET formation; Peptidyl-arginine Peptidyl-citrulline 2) Regulation of gene expression; 3) Activation of certain subroutines of apoptosis; 4) Terminal epidermal differentiation. D Gene Expression in the human body PAD1 All living layers of the epidermis, hair follicle, uterus, stomach Skeletal muscle, salivary gland, brain, immune cells, bone marrow, skin, PAD2 peripheral nerves, uterus, spleen, secretory gland, pancreas, kidney, inner ear C Conditions associated with PAD3 Hair follicle, skin, peripheral nerves excessive citrulination: PAD4 Immune cells, brain, uterus, joints, bone marrow 1) Rheumatoid arthritis; PAD6 Ovary, egg cells, embryo, testicle 2) Systemic lupus erythematosus; Determined substrates for citrullination 3) Peridontitis; Gene PAD1 4) Autoimmune encephalomyelitis; Keratin and filaggrin 5) Atherosclerosis; PAD2 Actin, Vimentin, Histones, Myelin basic protein 6) Thrombosis; PAD3 Vimentin, Filaggrin, Apoptosis-inducing factor 7) Multiple sclerosis. PAD4 Histones, Collagen type I, ING4, p300, p21, Lamin C, Nucleophosmin PAD6 ? © 2019 American Association for Cancer Research Figure 1. Summary of essential PADs/citrullination facts. A, Cartoon depicting PAD-mediated modification of arginine to citrulline residues. B, Lists of biological consequences of citrullination and key processes connected with this posttranslational modification. Information was obtained from refs. 1, 11, 22, 24, 27, 35, and 81. C, Medical conditions and diseases associated with pathologic citrullination. Information was obtained from refs. 1, 11, 22, 24, 27, 35, and 81. D, Tables explaining the tissue distribution of PADs and known substrates for citrullination. Information was obtained from refs. 1, 11, 22, 24, 27, 35, and 81. refs. 22, 23). They contain approximately 650 amino acids and Calcium concentration within the cell is maintained at low have a molecular weight of 74 kDa (22, 23). Substrate targets of levels, thus keeping PADs inactive under physiologic condi- þ different PADs are determined and partially overlapping tions (30). An increase in Ca2 levels leads to the activation of (Fig. 1D), but interestingly, not all arginine residues in a protein PADs; however, certain PAD-mediated processes such as gene can be citrullinated. For example, arginine residues in b-turns are regulation are described in conditions of physiologic concen- much more commonly citrullinated than those in a-helixes, trations of calcium, implying the existence of other unknown whereas location next to proline or glutamic acid substantially mechanism of PAD action (27). Apoptosis depends on high reduces the likelihood of arginine citrullination (24). The most cellular concentrations of calcium (31), and protein citrullina- frequent targets for PAD-driven citrullination are keratin, filag- tion is increased in apoptotic cells (30, 32). Cell terminal grin, vimentin, actin, histones, collagens, and myelin basic pro- epidermal differentiation is also dependent on high calcium tein (4, 5, 22), all of which having a high arginine content clearly concentrations accompanied by PAD-induced citrullination of important for their function (Fig. 1D). Notably, free arginine structural proteins such as Keratin (33). The function of PADs cannot