Citrullination in Cancer Arseniy E

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 2019 American Association for Cancer Research. expressed in a wide range of tissues and organs (Fig. 1D;

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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 ?

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 be citrullinated by PADs (22, 25). as activators and co-activators of transcriptional modulation In the cellular milieu, all the PADs are localized within the has also been documented (34, 35), and will be discussed in cytosol, but only PAD4 can additionally translocate to the nucleus the following sections of this review. Finally, calcium-inde- to citrullinate histones and transcription factors (owing to the pendent roles of PADs were reported; in particular, citrulli- presence of a canonical nuclear localization sequence in its nated proteins are more frequently subjected to degradation structure; refs. 26, 27). Citrullination of the extracellular matrix potentially due to conformation changes (16, 17), which may (ECM) also exists; for example, ECM deposition of PAD4 along additionally implicate PADs in facilitating the turnover of with extensive ECM protein citrullination were observed in colo- aberrant or redundant proteins. It is important to note that rectal cancer liver metastasis with exosomal route of PAD4 deliv- functional difference between PAD family members is still ery being proposed as a mechanism (28). Others, however, unstudied, yet PAD2 and PAD4 are the most broadly expressed speculated that extracellular citrullination can be potentiated by in humans with the latter being involved in several autoim- the leak of PADs during cell death (29). mune diseases.

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Prevalence of PADs in Tumor Tissues PAD4 was found to bind and subsequently citrullinate the inhibitor of growth 4 (ING4), another tumor suppressor protein Although it is difficult to accurately determine the extent of known to bind p53 (45). PAD4-driven citrullination of ING4 at overall citrullination in tissues and body fluids (36), measuring the nuclear localization sequence region prevented p53-to-ING4 the corresponding concentration of PAD enzymes appears to be a binding, repressed p53 acetylation, and subsequently inhibited robust way of estimating the frequency of this posttranslational downstream p21 expression (45). modification. PAD4 expression levels were increased in various Interaction of PAD4 with p53 was shown to citrullinate histone solid tumors relative to their respective normal counter- H4R3 upon chemotherapy treatment (46), with citrullinated parts (37, 38) as well as overrepresented in the peripheral blood regions being colocalized with decondensed soluble chromatin of patients with lung cancer (39). Approximately 40% of cells in in apoptotic cells, suggesting the direct involvement of the PAD4– malignant lymphomas also expressed PAD4, indicating that pp53 complex in apoptosis. PAD4 / mice displayed apoptosis expression of this protein is associated with cancer development resistance, whereas patients with lung cancer with citrullinated from all embryological lineages. Benign tumors and nontumor H4R3 expression had smaller tumor size compared with indivi- inflamed tissues did not express PAD4 (37), whereas metastasis duals with non-citrullinated H4R3 (46). exhibited much higher PAD4 levels compared with correspond- In another study (6), histone deacetylase 2 (HDAC2) was ing primary tumors (28), implicating citrullination in the pro- determined as a PAD4-interacting protein and they both were gression from benign neoplasm to invasive malignancy. Taken shown to bind to p53 and simultaneously associate with the p21 together, these data indicate that PAD4 is not uncommonly promoter in response to DNA damage in order to regulate gene overexpressed in tumors and thus may represent a biomarker or expression. In addition, inhibition of PAD4 and/or HDACs a putative therapeutic target for cancer treatment. impacted on histone modifications at p53 target gene promoters Although tumor-associated PAD2 was overexpressed in and reduced growth of cultured osteosarcoma cancer cells in a biopsies from patients with castration-resistant prostate cancer p53-dependent manner (6). Consistent with these reports, over- (CRPC; ref. 40), a downregulation of intertumoral PAD2 expres- expression of PAD4 in hematopoietic cancer cell lines increased sion was identified in a cohort of patient with colorectal cancer p53 and p21 expression and directly induced apoptosis (47). specimens in comparison with normal mucosa of healthy control Collectively, these studies point at the existence of versatile tissues (41). interactions between p53, proteins belonging to the p53 pathway, Because PADs 2 and 4 are the most broadly expressed in and PAD4. Interestingly, such relationship has not been described humans, other members of this family have not been extensively for other PADs, suggesting that PAD4 has a primary role in studied with regards to their abundance in cancer tissues. apoptosis regulation. RNA polymerase II (RNAP2) coordinates gene expression by Citrullination Affecting Cancer Cell influencing transcription elongation (48). Recently, PAD2 was shown to citrullinate RNAP2 in T47D breast cancer cells, activat- Signaling ing transcription of thousands of genes. Inhibition of citrullina- PADs and citrullination regulate gene transcription, mainly tion by Cl-amidine, PAD2 gene silencing, or mutation of R1810 acting as de-modifying factors to remove the histone (or any (site of RNAP2 citrullination) equally reduced cell proliferation other protein) methylation tag and converting the protein to its through the cell-cycle arrest at the G1 phase (49). Interestingly, basal state (42). Because DNA methylation is a key mediator of other members of the PAD family were unable to citrullinate many physiologic and pathologic conditions (43), replacing the RNAP2 and induce the above-mentioned effects (49). methyl group from arginine serves as powerful regulatory mech- Wnt/b-catenin signaling is important for carcinogenesis of anism controlled by PADs. As such, citrullination acts as on/off certain tumors, and PAD2-mediated citrullination was recently switch between different modes of transcriptional regulation, linked to this pathway (50). Antiparasitic drug temozolomide being particularly relevant for cancer, a disease strictly dependent (NTZ) inhibited Wnt/b-catenin signaling through directly target- on the activation of specific cell signaling programs. As an ing PAD2 as demonstrated by co-immunoprecipitation studies. apt example, after PAD4-driven citrullination of methylated Addition of NTZ resulted in a significant increase of b-catenin histone H3 Arg17, which is known to regulate estrogen-responsive citrullination by PAD2 and also substantially extended the half- genes, MCF-7 breast cancer cells displayed a drastic reduction in life of PAD2 protein, suggesting that NTZ enhances stability b-estradiol–induced genes, altering cell phenotype (42). None- of PAD2. Further, colony growth assays suggested that PAD2- theless, removal of methylation events by PADs remains largely mediated citrullination of b-catenin could limit the proliferation unstudied with only anecdotical experiments described in of colon cancer cells by inhibiting the Wnt pathway. As a result, literature. this elegant study uncovered a hitherto unrecognized mechanism Multiple interactions between PAD4 and p53 were reported, of Wnt signaling inhibition via PAD2. In accord, other studies suggesting the importance of PAD-induced citrullination in apo- independently proposed TMZ as a therapeutic target in colon ptosis. Expression of a major p53 target gene OKL38 was repressed adenocarcinoma, a malignancy characterized by frequent Wnt/ by a p53-mediated recruitment of PAD4 to the promoter of b-catenin deregulations (51, 52). OKL38 and subsequent removal of histone arginine methylation Recent evidence suggested the involvement of citrullination in mark, thus directly modulating apoptosis (44). In particular, Androgen signaling in prostate cancer (40). PAD2 was upregu- inhibition of PADs by their inhibitor Cl-amidine in breast and lated in patients with CRPC and overexpression of PAD2 by osteosarcoma cells resulted in OKL38 gene expression, and over- prostate cancer cells induced a CRPC-like phenotype in mouse expression of this gene in cell culture led to apoptosis and xenografts, which was mediated by androgen receptor signaling. mitochondria structural changes accompanied by release of cyto- In particular, PAD2-driven citrullination in the nucleus activated chrome c (44). the androgen receptor pathway, whereas PAD2 stabilized the

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androgen receptor and promoted its translocation to the nucleus. inhibitor gefitinib through an unknown mechanism (61). To Prostate cancer cells treated with PAD inhibitor Cl-amidine and conclude, more research should be done in order to elucidate PAD2-knockdown prostate cancer cells both exhibited a delay in the importance of citrullination in regulating the EMT plasticity. cancer progression in vitro and in vivo (40). The authors, however, did not explore which proteins and residues were citrullinated, thus providing little mechanistic explanation for the observed NETs Facilitating Cancer findings. Interestingly, PAD4 expression was shown to be induced NETs have been detected in several human cancer types by estrogen receptor signaling pathway in MCF-7 breast cancer (62–65), and some authors raised an interesting possibility of cells (53), indicating the complexity of the overall picture. their contribution to cancer development. Chronic inflammation In acute promyelocytic leukemia, mutated malignant cells fail had been suggested to awaken dormant malignant cells, but the to differentiate into healthy granulocytic cells and undergo mechanism remained elusive (66). Recently, in experimental lung uncontrolled self-renewal. A recent elegant study revealed that cancer model, inflammation-induced NETs were shown to awak- PAD4 expression is increased during the differentiation of HL-60 en dormant cancer cells through proteolytic remodeling of lam- leukemia cells induced by the administration of all-trans retinoic inin, followed by activation of Integrin signaling in tumor acid (ATRA) drug. This cell differentiation depended on the cells (67). In particular, NET-derived DNA acted as a proteolysis demethylation of the PAD4 promoter (54). Through citrullina- scaffold by releasing neutrophil elastase and MMP9, which in turn tion of histones, PAD4 stimulated the expression of hematopoi- cleaved laminin to generate biologically active epitopes (also etic transcription factors SOX4 and PU.1, which in turn promoted known as matrikines) driving cancer cell proliferation (67). These differentiation of HL-60 into granulocytic cells (54). This work findings indicate that chronic inflammation may provoke cancer highlighted the significance of arginine deimination in cell dif- recurrence after a long period of dormancy with NETs being a ferentiation, which is supported by a recent extensive study of driver of this process. In a different study, the presence of NETs in embryos where PAD4 and citrullination were established as major subcutaneous Lewis lung carcinoma (LLC) grafts increased tumor regulators of pluripotency (55). mass by 35% in comparison with PAD4 / mice in which tumors were devoid of NETs (68). Compared with LLC tumors, B16 grafts displayed much slower tumor growth, which was due to much Citrullination and Epithelial-to- fewer activated neutrophils. However, priming these neutrophils toward NETosis by the administration of G-CSF promoted NET Mesenchymal Transition generation along with an increase in B16 tumor growth (68). This TGFb signaling has been recognized as critical to cancer cell study nonetheless failed to provide a mechanistic explanation of invasion and metastasis through the activation of the epithelial- why NETs are required for cancer growth. to-mesenchymal transition (EMT; ref. 56). To this end, knock- Besides promoting tumor cell proliferation and growth, NETs down of PAD4 in breast cancer cells activated TGFb signaling via can also be important in metastatic cascade acting as an adhesion upregulation of Smad4, and induced EMT by reducing E-cadherin substrate for metastasizing cancer cells through Integrin bind- and promoting vimentin expression, further propelling cell inva- ing (69). Cultured tumor cells exhibited an augmented adhesion siveness (57). These effects were dependent on PAD4-driven to NETs, which was disrupted by pre-incubation with anti-integ- citrullination of transcription factor glycogen synthase kinase 3 rin antibodies (69). Furthermore, in a mouse model of sepsis, beta (GSK3b), which resulted in a translocation of GSK3b from microvascular NET deposition led to entrapment of circulating the cytoplasm into the nucleus and initiation of multiple gene lung carcinoma cells within DNA webs, contributing to develop- expression (57). In keeping with these findings, GSK3b was ment of metastases in vivo (70). This process was attenuated by reported to induce EMT (58) and control several transcription systemic administration of NET inhibitors DNAse or neutrophil factors related to cancer progression (59). Additionally, in a elastase inhibitors. The observed NET-mediated trapping of can- mouse model of hepatic metastases, pharmacological inhibition cer cells within hepatic sinusoids was associated with increased of PADs abated liver metastatic growth and enhanced the expres- formation of hepatic micrometastases at 48 hours and gross sion of mesenchymal markers, whereas the reverse process, mes- metastatic disease burden at 2 weeks following tumor cell injec- enchymal-to-epithelial transition (MET) was suggested to be of tion (70). Similar to the findings described in ref. 69, the authors importance in initiating metastatic colony formation (28). These demonstrated NETs entrapment of human and murine tumor studies collectively suggest that PAD4/citrullination axis might be cells in vitro (70). implicated in acquisition of prometastatic phenotype by tumors Conditions defining NET production in cancers is an intriguing through regulating EMT. question. Tohme and colleagues (65) demonstrated that isolated Consistent with data described above, in human triple-negative mouse neutrophils cultured in media conditioned by colon breast cancer (TNBC), PAD1 was shown to induce EMT through cancer cells generate NETs in hypoxic conditions characteristic suppressing ERK1/2 and P38 MAPK signaling through direct of surgical stress (e.g., cancer-associated hepatectomy). Surgically citrullination of MEK1 (60). This resulted in disrupting MEK1- stressed murine livers were prone to accommodate metastasizing mediated phosphorylation of ERK1/2, eventually leading to cells and give raise to colonies, whereas administration of NET MMP2 overexpression, eventually triggering EMT. inhibitors such as DNAse significantly reduced the onset and On the opposite, contradictory findings were provided by growth of metastases in surgically stressed but not intact murine Duan and colleagues (61), who reported that upregulation of livers. Similar results were shown upon the genetic or pharma- PAD4 can repress EMT through downregulating the expression cologic targeting of PAD4 in mice with experimental liver metas- of transcription factor Elk1 in lung cancer cell lines (HCC827 and tases. These results indicate that surgical stress followed by hyp- H1650). Interestingly, overexpression of PAD4 in HCC827 and oxia can serve as a requirement for NET generation in the tumor H1650 cells in this study inhibited the resistance of cells to EGFR microenvironment. Further mechanistic studies revealed that

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Table 1. Evidence summarizing the involvement of PADs and associated citrullination in cancer development and progression Subject of the study Aim of the study Major findings Ref. Prevalence of PADs in tumor tissues PAD4 The authors evaluated expression Having analyzed 1,673 clinical specimens, the authors found PAD4 to be (37) levels of PAD4 in various cancers by overexpressed in the carcinomas of the uterus, ovary, colon, bladder, IHC (33, 34) and qPCR (35) breast, liver, lung, endometrium, esophagus, kidney, and soft tissue in comparison with unaffected tissues. Benign and inflamed nontumor tissues showed no signs of PAD4 staining. A total of 162 of 167 individuals with esophageal cancer exhibited high or (38) moderate intratumoral PAD4 levels as evaluated by IHC. Sixteen healthy esophagus specimens showed no signs of PAD4 staining. A total of 100 subjects with non–small cell lung cancer demonstrated (39) significantly higher levels of PAD4 in the peripheral blood as compared with 100 healthy blood donors. PAD2 The authors examined the expression The authors found PAD2 to be overexpressed in 27 biopsy specimens from (40) of PAD4 in prostate cancer patients with CRPC as compared with 229 specimens from patients with specimens by IHC localized prostate cancer. PAD4 The authors studied the expression of The authors showed that hepatic metastases from five colon cancer liver (20) PAD4 in primary colon cancer and metastasis specimens exhibited higher PAD4 concentrations compared associated liver metastasis by with five adjacent unaffected livers, five primary colon cancer immunoblotting and quantitative specimens, or five unaffected colonic mucosas. proteomics PAD2 The authors evaluated expression Intratumoral PAD2 was downregulated in 98 colorectal cancer patient (41) levels of the PAD4 gene in colon specimens compared with normal mucosa of 50 healthy control tissues. cancer by PCR Citrullination affecting cancer cell signaling PAD4 The authors investigated citrullination Upon methylated histone H3 Arg17 citrullination, there was a drastic (42) of methylated histone H3 Arg17 in reduction in the expression of genes induced by b-estradiol through MCF-7 breast cancer cells histone H3 Arg17 methylation. PAD4 In MCF-7 breast cancer cells, the OKL38 was determined to be a p53 target gene, inducible by DNA (44) authors performed a DNA damage. Inhibition of PAD4 by Cl-amidine in MCF-7 and U2OS cells microarray analysis to identify genes resulted in the OKL38 gene expression, and overexpression of the regulated by PAD4 activity in cells OKL38 gene in cell culture led to apoptosis, mitochondria structure treated with Cl-amidine changes, and the delocalization of cytochrome c. Further research showed that OKL38 expression was induced by dynamic p53 and PAD4 binding and histone citrullination. PAD4 The authors searched for novel protein ING4 acted as a substrate for PAD4-induced citrullination. PAD4 (45) substrates of PAD4 in vitro citrullinated ING4 and could bind to ING4 regardless of calcium levels. PAD4-driven citrullination of ING4 at the nuclear localization sequence region prevented p53-to-ING4 binding, repressed p53 acetylation, and subsequently inhibited downstream p21 expression. Citrullination of ING4 promoted its degradation. PAD4 The authors studied the physiologic It was found that p53-PAD4 can citrullinate histone H4R3 in adriamycin- (46) role of p53-PAD4 interaction upon treated U2OS cells, and citrullination colocalized with decondensed the DNA damage using PAD4 soluble chromatin in apoptotic cells. PAD4-deficient mice exhibited deficient mice and clinical cancer resistance to apoptosis in response to gamma irradiation–induced DNA tissues damage. Patients with lung cancer with citrullinated H4R3 expression had smaller tumor size compared with individuals with non-citrullinated H4R3. PAD4 The authors investigated interactions HDAC2 was found to bind to PAD4 and they both were shown to bind to (6) between PAD4, HDAC2, and p53 in p53 and to associate with the p21 promoter in response to DNA damage U2OS cells to regulate expression of multiple genes. Inhibition of PAD4 and/or HDACs impacted on histone modifications at p53 target gene promoters and reduced growth of cultured osteosarcoma cancer cells in a p53-dependent manner. PAD4 The authors examined if inducible PAD4 overexpression induced apoptosis in HL-60 and Jurkat cells, and (47) overexpression of PAD4 enhances increased the expression of p53, p21, and Bax. apoptosis in vitro PAD2 The authors sought to explore the NTZ administration blocked Wnt/b-catenin signaling through stabilizing (50) antiparasitic drug NTZ in relation to PAD2, thus increasing the citrullination and turnover of b-catenin in Wnt/b-catenin signaling in multiple cancer cells. Colony growth assays showed that PAD2-mediated models citrullination of b-catenin limited proliferation of HCT116 and SW480 colon cancer cells by inhibiting the Wnt pathway. PAD2 The authors explored citrullination of In T47D breast cancer cells, PAD2 but not other members of PAD family (49) RNAP2 in vitro induced citrullination of the RNAP2 at the C-terminal domain R1810. This posttranslational modification substantially altered gene expression profile of cancer cells and impacted on their proliferation. (Continued on the following page)

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Table 1. Evidence summarizing the involvement of PADs and associated citrullination in cancer development and progression (Cont'd ) Subject of the study Aim of the study Major findings Ref. PAD2 The authors looked into PAD2 PAD2 was found to be overexpressed in patients with CRPC and showed a (40) expression in prostate cancer and response to androgen receptor. Levels of PAD4 and citrullinated protein investigated its interactions with the were higher in CRPC tissues than in the localized prostate cancer androgen receptor in vitro and in tissues. Higher levels of PAD2 were associated with elevated levels of vivo citrullinated protein. PAD2 stabilized the androgen receptor protein and facilitated its nuclear translocation. Combined inhibition of the androgen receptor protein and PAD2 synergistically delayed CRPC growth both in vitro and in vivo. PAD4 The authors were interested in the PAD4 expression gradually increased during the differentiation of (54) involvement of PAD4 in leukemia cells. PAD4 promoter underwent demethylation during HL-60 differentiation of leukemia cells differentiation induced by ATRA. PAD4 regulated SOX4 expression through histone citrullination and exerted activity in a SOX4-dependent manner. SOX4, in turn, mediated the regulation of PU.1, which ultimately stimulated differentiation of HL-60 into granulocytic cells. Citrullination and EMT PAD4 The authors utilized an in vitro gene The knockdown of PAD4 in MCF-7 breast cancer cells activated TGFb (57) silencing model to study the effect signaling via Smad4 and p-Smad2, thus promoting the EMT by reducing of PAD4 on TGFb signaling and EMT E-cadherin and promoting vimentin, ultimately stimulating cell invasiveness. This was dependent on PAD4-mediated citrullination of transcription factor GSK3b, resulting in a translocation of GSK3b from the cytoplasm into the nucleus and triggering gene expression. PAD4 The authors treated mice in an Treatment with BB-Cl-amidine reduced metastatic burden in mice. (28) experimental liver metastasis Metastases explanted from untreated animals exhibited high experiment with the PAD inhibitor citrullination, upregulated epithelial markers (E-cadherin, cytokeratin 7, BB-Cl-amidine and tight junction protein-1) and downregulated mesenchymal markers ZEB1 and N-cadherin. BB-Cl-amidine-treated mice exhibited the reverse, with decreased expression of epithelial markers and increased expression of mesenchymal markers. PAD1 The authors explored the molecular PAD1 expression was upregulated in patients with TNBC based on (60) mechanisms through which PAD1 bioinformatics studies. Knockdown or silencing of PAD1 reduced MDA- affects TNBC invasion using the MB-231 cancer cell proliferation, migration, and invasion in vitro. MDA-MB-231 cell line Further, silencing of PAD1 reduced MMP expression and reversed the EMT in vitro through direct citrullination of MEK1. PAD4 The authors investigated EMT changes Both mRNA and protein levels of Elk1, N-cadherin, and vimentin were (61) upon the transfection of lung cancer substantially downregulated, whereas E-cadherin and a-catenin were cells HCC827 and H1650 by pCMV- upregulated in vitro upon transfection with PAD4 cDNA. In concert, 2a/2b-PAD4 transfection or used addition of PAD4 siRNA to cells had a reverse impact in comparison with siRNA to silence PAD4. pCMV-2a/2b-PAD4 transfection. In addition, overexpression of PAD4 inhibited the resistance of cells to EGFR inhibitor gefitinib. NETs facilitating cancer PAD4 and NETs Using multiple models, the authors NETs promoted dormant cancer cell awakening in mice after sustained (67) tested if NETs formed during lung lung inflammation caused by lipopolysaccharide inhalation or tobacco inflammation could induce smoke exposure. NETs also induced dormant cancer cell awakening in awakening of dormant metastatic vitro in the absence of other host cells. Mechanistically, NET-derived cells. DNA acted as a proteolysis scaffold by releasing neutrophil elastase and MMP9, which in turn cleaved laminin to generate biologically active epitopes driving cancer cell proliferation PAD4 and NETs Using PAD4/ mice, the authors Unlike NET-free PAD4/ mice, the presence of NETs in wild-type animals (68) addressed the role of the host led to an increased tumor growth of LLC grafts. B16 melanoma PAD4/NETs in tumor development. subcutaneous tumor growth was not affected by the host PAD4 deficiency due to fewer cancer-associated neutrophils. Priming of neutrophils toward NETosis by G-CSF administration in mice promoted NET formation in the B16 tumors along with an increase in tumor growth. NETs The authors performed adhesion Cultured tumor cells U-87 MG, HT-1080, DU 145, PC-3, H1975 exhibited an (69) assays for different cancer cells augmented adhesion to NETs, which was disrupted by pre-incubation using isolated NETs as an adhesion with anti-integrin antibodies. There was no effect for A-431 cells in terms substrate. of adhesion to NETs. NETs The authors hypothesized that upon In a mouse model of sepsis (cecal ligation and puncture), the authors (70) severe infection, NETs are able to observed accumulation of NETs in the liver and lung. Systemic sepsis trap circulating cancer cells and to facilitated hepatic metastasis formation in mice, which was attenuated promote their early adhesion in by administration of DNAse or neutrophil elastase inhibitors. The distant organ sites observed metastasis formation was due to cancer cell entrapment by NETs within hepatic sinusoids. The NET trapping correlated with increased formation of hepatic micrometastases at 48 hours and gross metastatic disease burden at 2 weeks following tumor cell injection. NET entrapment of human and murine tumor cells in vitro was also observed. (Continued on the following page)

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Table 1. Evidence summarizing the involvement of PADs and associated citrullination in cancer development and progression (Cont'd ) Subject of the study Aim of the study Major findings Ref. NETs The authors hypothesized that upon Hepatic resection resulted in liver NET formation based on clinical sample (65) the surgical stress, NETs can be assessment. Liver hypoxic microenvironment associated with hepatic formed in the liver and may promote resection stimulated NET generation. Surgical stress facilitated liver the adhesion of circulating tumor metastasis in mice, whereas administration of a NET inhibitor DNAse or cells and enable metastatic growth genetic targeting of PAD4 diminished tumor growth. NETs were shown in mice to induce protumorigenic effects through activation of TLR9 pathway. NETs The authors investigated a possible Analysis of clinical samples showed that nonalcoholic steatohepatitis was (72) link between NETs and nonalcoholic characterized by neutrophil infiltration and NET deposition, as well as steatohepatitis in vivo, in vitro,and proinflammatory response. Inhibition of NETs in mice reduced hepatic analysing clinical specimens inflammation and reduced liver cancer progression. In addition, free fatty acids elevated in nonalcoholic steatohepatitis were shown to stimulate NET formation in vitro. NETs The authors explored the prognostic Intratumoral NETs predicted poor postsurgical survival in 317 patients with (73) significance of tumor-infiltrating pancreatic cancer. Multivariate analysis identified NETs as an NETs in pancreatic ductal independent prognostic factor for overall and recurrence-free survival. adenocarcinoma patients Citrullination induces antitumor immunity citrullinated a-enolase peptides The authors questioned whether The authors identified citrullinated a-enolase peptides, which induced a (76) citrullinated peptides can induce an Th1 response in mice as assessed by granzyme B and IFNg. Vaccination antitumor immune response in vivo against the citrullinated a-enolase peptide resulted in a strong antitumor effect in melanoma, pancreatic, and lung subcutaneous cancer models in vivo. A T-cell repertoire to citrullinated a-enolase peptides was also identified in humans. Citrullinated vimentin peptides The authors questioned if citrullinated Immunization of tumor-bearing mice with citrullinated vimentin peptides (77) peptides can elicit an antitumor resulted in effective antitumor responses against B16F1 grafts, which immune response in vivo were CD4- but not CD8-mediated and also IFNg dependent. Immunized mice demonstrated 80% survival on the 40th day after a single dose of citrullinated vimentin peptides injected on day 10 posttumor initiation. Citrullination as a biomarker citrullinated histone H3 The authors sought to define the levels Cancer patients had a threefold increase in the median level of citrullinated (79) of circulating citrullinated histone H3 histone H3 in comparison with healthy individuals and severely ill in patients with advanced cancer patients without known cancer. Blood concentrations of citrullinated histone H3 correlated with activation of neutrophils in patients with advanced cancer. In cancer patients, higher levels of serum citrullinated histone H3 were prognostic for short-term mortality.

NETs induced protumorigenic effects through a release of high in a substantial survival advantage in subcutaneous tumor models mobility group box 1 (HMGB1) protein, which in turn activated of melanoma, pancreatic, and lung carcinoma (76). Vaccinated þ Toll-like receptor 9 (TLR9) signaling pathway in cancer cells, mice demonstrated a stronger CD4 T-cell response as assessed by eventually promoting their proliferation (65). Consistent with IFNg and granzyme B levels. Interestingly, healthy individuals þ these findings, hypoxia induced generation of citrullinated pro- exhibited a repertoire of CD4 T cells capable of responding to the teins in malignant glioma cells (71). Apart from hypoxia, elevated citrullinated a-enolase peptide. free fatty acids were also reported to stimulate NET formation In a similar way, Brentville and colleagues (77) vaccinated concurrently promoting hepatocellular carcinoma growth in mice previously challenged with subcutaneous B16 melanoma mice (72). tumors with citrullinated vimentin peptide, observing an effec- Overall, these findings link PAD/citrullination-mediated for- tive anticancer response with 80% of mice surviving after 50 mation of NETs with tumor growth and metastatic spreading of days of the experiment. Administration of blocking anti-CD4 cancer cells potentially through ECM remodeling, surgical stress, but not anti-CD8 antibodies concurrently with citrullinated hypoxia, increased fatty acid levels, or through physically binding vimentin peptide negated the survival advantage, indicating þ metastasizing cells to establish colonies. In support of these basic that the observed effect was CD4 T-cell dependent. This is studies results, tumor-infiltrating NETs predicted poor postsurgi- in concert with findings showing a more pronounced antitu- cal survival of pancreatic cancer patients (73), emphasizing the mor response to CD4 neo-epitopes in comparison with clinical relevance of this phenomenon. CD8 neo-epitopes (78). Collectively, these reports testify to the feasibility of the use of citrullinated peptides as boosters of antitumor immunity and Citrullination Induces Antitumor Immunity combinations of selected citrullinated peptides in principle may Citrullinated epitopes are presented on MHC-II and stimulate be utilized to target certain cancers. þ CD4 T-cell response (74). Because citrullination also intensifies the autoimmune response in RA and SLE, attempts have been made to utilize citrullinated proteins as vaccines to propel the Citrullination as a Biomarker antitumor immunity. In mice bearing subcutaneous tumors, Recently, citrullinated histone was proposed as a cancer immunization with citrullinated peptides of a-enolase (a key biomarker in a study where 60 patients with different malig- enzyme responsible for Warburg effect in cancers; ref. 75) resulted nancies of advanced stage exhibited a three-fold increase in

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Altering expression of Inhibition of CRPC-like thousands of genes and Wnt/β-catenin phenotype in affecting proliferation signaling PC cells

β-catenin c Androgen RNAP2 c receptor +NTZ Stabilization and PAD2 nuclear translocation

MEK1 PAD1 MEK1 c EMT ERK1/2 MMP2

PAD4 Figure 2. Schematic illustrating how PAD-mediated citrullination may affect cell signaling to facilitate cancer progression. PC, prostate cancer. p53 c c ING4 Histones GSK3β c c

p53 ING4 Differentiation EMT SOX4 E-cadherin PU.1 (SPI1) Estrogen signaling Vimentin Regulation p53 of apoptosis ac

Interaction/promotion Inhibition/blockingc Citrullination ac Acetylation

the mean concentration of serum citrullinated histone H3 in Synopsis comparison with 50 healthy individuals or 51 severely ill Posttranslational modifications of human proteome have a patients without known cancer (79). These data stipulated major impact on health and disease, expanding the functional that cancer patients have elevated serum levels of citrullinated diversity of proteins by adding new functional groups. In cancers, proteins and that circulating citrullinated histone H3 is not many physiologically relevant modifications including citrullina- characteristic of general disease burden. In a cohort of cancer tion have been infrequently studied thus leaving a potentially patients, invasive tumors had on average more serum citrul- important avenue for research. Evidence summarized in this linated histone H3 than localized cancers, in agreement with review implicates citrullination in facilitating cancer development previous reports linking PADs and associated citrullination to through several distinct mechanisms (Table 1; Fig. 2). First, metastasis (70). Of note, plasma levels of citrullinated his- citrullination of histones or transcription factors with subsequent tone H3 correlated with higher levels of cell-free DNA and removal of methylation tag dramatically influences fundamental neutrophil activation in cancer patients, alluding on the cellular processes such as apoptosis and differentiation. It also involvement of NETs. Strikingly, the authors found a strong affects pathways directly contributing to cancer progression, spe- correlation between higher plasma levels (>29.8 ng/mL, cifically the Wnt and androgen receptor signaling pathways. It above the 75th percentile) of citrullinated histone H3 and remains unclear, however, what mechanism is in control of such two-fold increased risk of short-term mortality (79). Because PAD-driven demethylation, whether citrullination of met-Arg RA is diagnosed by antibody responses to citrullinated pro- residues occurs constantly during the cell cycle or it is entirely teins (4), it would be of great interest to examine whether context dependent, and if this phenomenon prevails at particular clinically used anti-cyclic citrullinated peptide antibodies are stages of metastatic development or in specific regions of the successful in detecting malignant neoplasms in prospective or primary tumor or metastasis. retrospective cohorts.

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CI Interaction/ NH promotion PADs HN Inhibition/ Cl-amidine blocking O c Citrullination N Histones H N c HN + Hypoxia + Surgical stress + Free fatty acids

Figure 3. Graphical demonstration of how PAD-driven citrullination of NET histones promotes NET formation, which in turn facilitate tumor growth and metastasis. HMGB1 release

Physical Neutrophil elastase- entrapment of and MMP9- cancer cells mediated laminin (Integrin-mediated) cleavage Tumor growth TLR9 Metastasis Dormant cell awakening

Second, amplification of T-cell immunity by citrullinated anti- viability of normal cells (A.E. Yuzhalin; submitted for publica- gens, specifically cit-a-enolase and cit-vimentin, may potentially tion; ref. 81), potentially opening avenues for targeted therapy. be utilized in cancer immunotherapy. Third, there is some pre- Another interesting future direction could be detecting the titers of liminary data suggesting that PADs and associated citrullination anti-citrullinated peptide antibodies in cohorts of cancer patients. are able to enforce EMT thereby contributing to metastasis. Having said that, I understand that repertoire of biological func- Fourth, NETs can also facilitate tumor growth and metastasis tions mediated by citrullination is clearly more diverse than we seeding via multiple mechanisms, including the entrapment of think, and additional basic research should be further performed circulating cancer cells at distant sites and awakening of dormant to clarify the molecular basis underlying the influence of PADs cells through cleaved laminin peptides (Fig. 3). Finally, serum and associated deimination on tumor progression. levels of citrullinated peptides and cell-free DNA characteristic of NETs were proposed as novel biomarkers of solid tumors, which is fl in line with the fact that many cancers display elevated intratu- Disclosure of Potential Con icts of Interest No potential conflicts of interest were disclosed. moral levels of PAD4. Taken together, these data justify the feasibility of testing PAD Acknowledgments inhibitors in preclinical models of cancer. Several irreversible This work was not financially supported by any funding source. inhibitors for these enzymes, including Cl-amidine, have proved their efficacy in multiple in vitro and in vivo experiments (14, 28, 80). Importantly, pharmacologic inhibition of PADs substantially Received September 5, 2018; revised December 9, 2018; accepted January 11, reduced proliferation of cancer cells while not affecting the 2019; published first March 20, 2019.

References 1. Valesini G, Gerardi MC, Iannuccelli C, Pacucci VA, Pendolino M, Shoenfeld 5. Lee C-Y, Wang D, Wilhelm M, Zolg DP, Schmidt T, Schnatbaum K, et al. Y. Citrullination and autoimmunity. Autoimmun Rev 2015;14:490–7. Mining the human tissue proteome for protein citrullination. Mol Cell 2. Aratani S, Fujita H, Yagishita N, Yamano Y, Okubo Y, Nishioka K, et al. Proteomics 2018;17:1378–91. Inhibitory effects of ubiquitination of synoviolin by PADI4. Mol Med Rep 6. Li P, Wang D, Yao H, Doret P, Hao G, Shen Q, et al. Coordination of PAD4 2017;16:9203–9. and HDAC2 in the regulation of p53-target gene expression. Oncogene 3. Travers TS, Harlow L, Rosas IO, Gochuico BR, Mikuls TR, Bhattacharya SK, 2010;29:3153–62. et al. Extensive citrullination promotes immunogenicity of HSP90 through 7. Hagiwara T, Nakashima K, Hirano H, Senshu T, Yamada M. protein unfolding and exposure of cryptic epitopes. J Immunol 2016;197: Deimination of arginine residues in nucleophosmin/B23 and his- 1926–36. tones in HL-60 granulocytes. Biochem Biophys Res Commun 2002; 4. Tilvawala R, Nguyen SH, Maurais AJ, Nemmara VV, Nagar M, Salinger AJ, 290:979–83. et al. The rheumatoid arthritis-associated citrullinome. Cell Chem Biol 8. Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, Gizinski A, 2018;25:691–704.e6. Yalavarthi S, Knight JS, et al. NETs are a source of citrullinated

www.aacrjournals.org Cancer Res; 79(7) April 1, 2019 OF9

Yuzhalin

autoantigens and stimulate inflammatory responses in rheumatoid 30. Asaga H, Yamada M, Senshu T. Selective deimination of vimentin in arthritis. Sci Transl Med 2013;5:178ra40. calcium ionophore-induced apoptosis of mouse peritoneal macrophages. 9. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Biochem Biophys Res Commun 1998;243:641–6. Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science 31. Mattson MP, Chan SL. Calcium orchestrates apoptosis. Nat Cell Biol 2003; 2004;303:1532–5. 5:1041–3. 10. Li P, Li M, Lindberg MR, Kennett MJ, Xiong N, Wang Y. PAD4 is essential for 32. Hsu P-C, Liao Y-F, Lin C-L, Lin W-H, Liu G-Y, Hung H-C. Vimentin is antibacterial innate immunity mediated by neutrophil extracellular traps. involved in peptidylarginine deiminase 2-induced apoptosis of activated J Exp Med 2010;207:1853–62. Jurkat cells. Mol Cells 2014;37:426–34. 11. Darrah E, Andrade F. Rheumatoid arthritis and citrullination. Curr Opin 33. Senshu T, Kan S, Ogawa H, Manabe M, Asaga H. Preferential deimination Rheumatol 2018;30:72–8. of keratin K1 and filaggrin during the terminal differentiation of human 12. Konig MF, Abusleme L, Reinholdt J, Palmer RJ, Teles RP, Sampson K, et al. epidermis. Biochem Biophys Res Commun 1996;225:712–9. Aggregatibacter actinomycetemcomitans-induced hypercitrullination 34. Lee Y-H, Coonrod SA, Kraus WL, Jelinek MA, Stallcup MR. Regulation of links periodontal infection to autoimmunity in rheumatoid arthritis. coactivator complex assembly and function by protein arginine methyl- Sci Transl Med 2016;8:369ra176. ation and demethylimination. Proc Natl Acad Sci U S A 2005;102:3611–6. 13. Carrillo-Vico A, Leech MD, Anderton SM. Contribution of myelin auto- 35. Wang S, Wang Y. Peptidylarginine deiminases in citrullination, gene antigen citrullination to T cell autoaggression in the central nervous system. regulation, health and pathogenesis. Biochim Biophys Acta 2013;1829: J Immunol 2010;184:2839–46. 1126–35. 14. Knight JS, Subramanian V, O'Dell AA, Yalavarthi S, Zhao W, Smith CK, et al. 36. Hensen SMM, Pruijn GJM. Methods for the detection of peptidylarginine Peptidylarginine deiminase inhibition disrupts NET formation and pro- deiminase (PAD) activity and protein citrullination. Mol Cell Proteomics tects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. 2014;13:388–96. Ann Rheum Dis 2015;74:2199–206. 37. Chang X, Han J, Pang L, Zhao Y, Yang Y, Shen Z. Increased PADI4 15. Caprariello A V, Rogers JA, Morgan ML, Hoghooghi V, Plemel JR, Koebel A, expression in blood and tissues of patients with malignant tumors. et al. Biochemically altered myelin triggers autoimmune demyelination. BMC Cancer 2009;9:40. doi: 10.1186/1471-2407-9-40. Proc Natl Acad Sci U S A 2018;115:5528–33. 38. Chang X, Hou X, Pan J, Fang K, Wang L, Han J. Investigating the pathogenic 16. Pritzker LB, Joshi S, Gowan JJ, Harauz G, Moscarello MA. Deimination of role of PADI4 in oesophageal cancer. Int J Biol Sci 2011;7:769–81. myelin basic protein. 1. Effect of deimination of arginyl residues of myelin 39. Ulivi P, Mercatali L, Casoni G-L, Scarpi E, Bucchi L, Silvestrini R, et al. basic protein on its structure and susceptibility to digestion by cathepsin D. Multiple marker detection in peripheral blood for NSCLC diagnosis. Biochemistry 2000;39:5374–81. PLoS One 2013;8:e57401. 17. Moscarello MA, Mastronardi FG, Wood DD. The role of citrullinated 40. Wang L, Song G, Zhang X, Feng T, Pan J, Chen W, et al. PADI2-mediated proteins suggests a novel mechanism in the pathogenesis of multiple citrullination promotes prostate cancer progression. Cancer Res 2017;77: sclerosis. Neurochem Res 2007;32:251–6. 5755–68. 18. Knight JS, Luo W, O'Dell AA, Yalavarthi S, Zhao W, Subramanian V, et al. 41. Cantarino N, Musulen E, Valero V, Peinado MA, Perucho M, Moreno V, Peptidylarginine deiminase inhibition reduces vascular damage and mod- et al. Downregulation of the deiminase PADI2 is an early event in colorectal ulates innate immune responses in murine models of atherosclerosis. carcinogenesis and indicates poor prognosis. Mol Cancer Res 2016;14: Circ Res 2014;114:947–56. 841–8. 19. Savchenko AS, Inoue A, Ohashi R, Jiang S, Hasegawa G, Tanaka T, et al. 42. Wang Y, Wysocka J, Sayegh J, Lee Y-H, Perlin JR, Leonelli L, et al. Human Long pentraxin 3 (PTX3) expression and release by neutrophils in vitro and PAD4 regulates histone arginine methylation levels via demethylimina- in ulcerative colitis. Pathol Int 2011;61:290–7. tion. Science 2004;306:279–83. 20. Chumanevich AA, Causey CP, Knuckley BA, Jones JE, Poudyal D, Chu- 43. Robertson KD, Wolffe AP. DNA methylation in health and disease. Nat Rev manevich AP, et al. Suppression of colitis in mice by Cl-amidine: a novel Genet 2000;1:11–9. peptidylarginine deiminase inhibitor. AJP Gastrointest Liver Physiol 2011; 44. Yao H, Li P, Venters BJ, Zheng S, Thompson PR, Pugh BF, et al. Histone Arg 300:G929–38. modifications and p53 regulate the expression of OKL38, a mediator of 21. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell apoptosis. J Biol Chem 2008;283:20060–8. 2011;144:646–74. 45. Guo Q, Fast W. Citrullination of inhibitor of growth 4 (ING4) by pepti- 22. Witalison EE, Thompson PR, Hofseth LJ. Protein arginine deiminases and dylarginine deminase 4 (PAD4) Disrupts the interaction between ING4 associated citrullination: physiological functions and diseases associated and p53. J Biol Chem 2011;286:17069–78. with dysregulation. Curr Drug Targets 2015;16:700–10. 46. Tanikawa C, Espinosa M, Suzuki A, Masuda K, Yamamoto K, Tsuchiya E, 23. Rohrbach AS, Arandjelovic S, Mowen KA. Physiological pathways of PAD et al. Regulation of histone modification and chromatin structure by the activation and citrullinated epitope generation. In: Nicholas A, Bhatta- p53-PADI4 pathway. Nat Commun 2012;3:676. doi: 10.1038/ncomms1676. charya S, editors. Protein deimination in human health and disease. New 47. Liu G-Y, Liao Y-F, Chang W-H, Liu C-C, Hsieh M-C, Hsu P-C, et al. York, NY: Springer New York; 2014. p. 1–24. Overexpression of peptidylarginine deiminase IV features in apoptosis of 24. Gyorgy€ B, Toth E, Tarcsa E, Falus A, Buzas EI. Citrullination: a posttrans- haematopoietic cells. Apoptosis 2006;11:183–96. lational modification in health and disease. Int J Biochem Cell Biol 2006; 48. Young RA. RNA polymerase II. Annu Rev Biochem 1991;60:689–715. 38:1662–77. 49. Sharma P, Lioutas A, Fernandez-Fuentes N, Quilez J, Carbonell-Caballero J, 25. Kearney PL, Bhatia M, Jones NG, Yuan L, Glascock MC, Catchings KL, et al. Wright RHG, et al. Arginine citrullination at the C-terminal domain Kinetic characterization of protein arginine deiminase 4: a transcriptional controls RNA polymerase II transcription. Mol Cell 2019;73:84–96.e7. corepressor implicated in the onset and progression of rheumatoid arthri- 50. Qu Y, Olsen JR, Yuan X, Cheng PF, Levesque MP, Brokstad KA, et al. Small tis. Biochemistry 2005;44:10570–82. molecule promotes b-catenin citrullination and inhibits Wnt signaling in 26. Nakashima K, Hagiwara T, Yamada M. Nuclear localization of peptidy- cancer. Nat Chem Biol 2017;14:94–101. larginine deiminase V and histone deimination in granulocytes. J Biol 51. Muller€ J, Sidler D, Nachbur U, Wastling J, Brunner T, Hemphill A. Thia- Chem 2002;277:49562–8. zolides inhibit growth and induce glutathione- S -transferase Pi (GSTP1)- 27. Vossenaar ER, Zendman AJW, Van Venrooij WJ, Pruijn GJM. PAD, a dependent cell death in human colon cancer cells. Int J Cancer 2008;123: growing family of citrullinating enzymes: genes, features and involvement 1797–806. in disease. BioEssays 2003;25:1106–18. 52. Senkowski W, Zhang X, Olofsson MH, Isacson R, Hoglund€ U, Gustafsson 28. Yuzhalin AE, Gordon-Weeks AN, Tognoli ML, Jones K, Markelc B, M, et al. Three-dimensional cell culture-based screening identifies the Konietzny R, et al. Colorectal cancer liver metastatic growth depends on anthelmintic drug nitazoxanide as a candidate for treatment of colorectal PAD4-driven citrullination of the extracellular matrix. Nat Commun 2018; cancer. Mol Cancer Ther 2015;14:1504–16. 9:4783. doi: 10.1038/s41467-018-07306-7. 53. Dong S, Zhang Z, Takahara H. Estrogen-enhanced peptidylarginine dei- 29. Skriner K, Adolph K, Jungblut PR, Burmester GR. Association of citrulli- minase type IV gene (PADI4) expression in MCF-7 cells is mediated by nated proteins with synovial exosomes. Arthritis Rheum 2006;54: estrogen receptor-a-promoted transfactors activator protein-1, nuclear 3809–14. factor-Y, and Sp1. Mol Endocrinol 2007;21:1617–29.

OF10 Cancer Res; 79(7) April 1, 2019 Cancer Research

Citrullination in Cancer

54. Song G, Shi L, Guo Y, Yu L, Wang L, Zhang X, et al. A novel PAD4/SOX4/ 68. Demers M, Wong SL, Martinod K, Gallant M, Cabral JE, Wang Y, et al. PU.1 signaling pathway is involved in the committed differentiation of Priming of neutrophils toward NETosis promotes tumor growth. Oncoim- acute promyelocytic leukemia cells into granulocytic cells. Oncotarget munology 2016;5:e1134073. 2016;7:3144–57. 69. Monti M, De Rosa V, Iommelli F, Carriero MV, Terlizzi C, Camerlingo R, 55. Christophorou MA, Castelo-Branco G, Halley-Stott RP, Oliveira CS, Loos R, et al. Neutrophil extracellular traps as an adhesion substrate for different Radzisheuskaya A, et al. Citrullination regulates pluripotency and histone tumor cells expressing RGD-binding integrins. Int J Mol Sci 2018;19. H1 binding to chromatin. Nature 2014;507:104–8. doi: 10.3390/ijms19082350. 56. David CJ, Huang Y-H, Chen M, Su J, Zou Y, Bardeesy N, et al. TGF-b tumor 70. Cools-Lartigue J, Spicer J, McDonald B, Gowing S, Chow S, Giannias B, et al. suppression through a lethal EMT. Cell 2016;164:1015–30. Neutrophil extracellular traps sequester circulating tumor cells and pro- 57. Stadler SC, Vincent CT, Fedorov VD, Patsialou A, Cherrington BD, Waksh- mote metastasis. J Clin Invest 2013;123:3446–58. lag JJ, et al. Dysregulation of PAD4-mediated citrullination of nuclear 71. Sase T, Arito M, Onodera H, Omoteyama K, Kurokawa MS, Kagami Y, et al. GSK3b activates TGF-b signaling and induces epithelial-to-mesenchymal Hypoxia-induced production of peptidylarginine deiminases and citrulli- transition in breast cancer cells. Proc Natl Acad Sci U S A 2013;110: nated proteins in malignant glioma cells. Biochem Biophys Res Commun 11851–6. 2017;482:50–6. 58. Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, et al. Dual regulation of 72. van der Windt DJ, Sud V, Zhang H, Varley PR, Goswami J, Yazdani HO, et al. Snail by GSK-3b-mediated phosphorylation in control of epithelial–mes- Neutrophil extracellular traps promote infl ammation and development of enchymal transition. Nat Cell Biol 2004;6:931–40. hepatocellular carcinoma in nonalcoholic steatohepatitis. Hepatology 59. Xu C, Kim N-G, Gumbiner BM. Regulation of protein stability by GSK3 2018;68:1347–60. mediated phosphorylation. Cell Cycle 2009;8:4032–9. 73. Jin W, Xu H-X, Zhang S-R, Li H, Wang W-Q, Gao H-L, et al. Tumor- 60. Qin H, Liu X, Li F, Miao L, Li T, Xu B, et al. PAD1 promotes epithelial- infiltrating NETs predict postsurgical survival in patients with pancreatic mesenchymal transition and metastasis in triple-negative breast cancer ductal adenocarcinoma. Ann Surg Oncol 2019;26:635–43. cells by regulating MEK1-ERK1/2-MMP2 signaling. Cancer Lett 2017;409: 74. Ireland JM, Unanue ER. Autophagy in antigen-presenting cells results in 30–41. presentation of citrullinated peptides to CD4 T cells. J Exp Med 2011;208: 61. Duan Q, Pang C, Chang N, Zhang J, Liu W. Overexpression of PAD4 2625–32. suppresses drug resistance of NSCLC cell lines to gefitinib through inhibit- 75. Capello M, Ferri-Borgogno S, Riganti C, Chattaragada MS, Principe M, ing Elk1-mediated epithelial-mesenchymal transition. Oncol Rep 2016;36: Roux C, et al. Targeting the Warburg effect in cancer cells through ENO1 551–8. knockdown rescues oxidative phosphorylation and induces growth arrest. 62. Yang C, Sun W, Cui W, Li X, Yao J, Jia X, et al. Procoagulant role of Oncotarget 2016;7:5598–612. neutrophil extracellular traps in patients with gastric cancer. Int J Clin Exp 76. Cook K, Daniels I, Symonds P, Pitt T, Gijon M, Xue W, et al. Citrullinated Pathol 2015;8:14075–86. a-enolase is an effective target for anti-cancer immunity. Oncoimmunol- 63. Berger-Achituv S, Brinkmann V, Abed UA, Kuhn€ LI, Ben-Ezra J, Elhasid ogy 2018;7:e1390642. R, et al. A proposed role for neutrophil extracellular traps in cancer 77. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, immunoediting. Front Immunol 2013;4:48. doi: 10.3389/fimmu. et al. Citrullinated vimentin presented on MHC-II in tumor cells is a target 2013.00048. for CD4þ T-cell-mediated antitumor immunity. Cancer Res 2016;76: 64. Merza M, Hartman H, Rahman M, Hwaiz R, Zhang E, Renstrom€ E, et al. 548–60. Neutrophil extracellular traps induce trypsin activation, inflammation, and 78. Kreiter S, Vormehr M, van de Roemer N, Diken M, Lower€ M, Diekmann J, tissue damage in mice with severe acute pancreatitis. Gastroenterology et al. Mutant MHC class II epitopes drive therapeutic immune responses to 2015;149:1920–31.e8. cancer. Nature 2015;520:692–6. 65. Tohme S, Yazdani HO, Al-Khafaji AB, Chidi AP, Loughran P, Mowen K, 79. Thalin C, Lundstrom€ S, Seignez C, Daleskog M, Lundstrom€ A, Henriksson et al. Neutrophil extracellular traps promote the development and pro- P, et al. Citrullinated histone H3 as a novel prognostic blood marker in gression of liver metastases after surgical stress. Cancer Res 2016;76: patients with advanced cancer. PLoS One 2018;13:e0191231. 1367–80. 80. Kawalkowska J, Quirke A-M, Ghari F, Davis S, Subramanian V, Thompson 66. Manjili MH. Tumor dormancy and relapse: from a natural byproduct of PR, et al. Abrogation of collagen-induced arthritis by a peptidyl arginine evolution to a disease state. Cancer Res 2017;77:2564–9. deiminase inhibitor is associated with modulation of T cell-mediated 67. Albrengues J, Shields MA, Ng D, Park CG, Ambrico A, Poindexter ME, et al. immune responses. Sci Rep 2016;6:26430. doi: 10.1038/srep26430. Neutrophil extracellular traps produced during inflammation awaken 81. Slack JL, Causey CP, Thompson PR. Protein arginine deiminase 4: a target dormant cancer cells in mice. Science 2018;361:eaao4227. for an epigenetic cancer therapy. Cell Mol Life Sci 2011;68:709–20.

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Citrullination in Cancer

Arseniy E. Yuzhalin

Cancer Res Published OnlineFirst March 20, 2019.

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