Abstract
Posttranslational modifications of proteins have been implicated in pathogenesis of numerous diseases. Arginine deimination (also known as citrullination) has a principal role in progression of rheumatoid arthritis through generation of autoantibodies and exacerbation of the inflammatory response. Recently, multiple research groups provided solid evidence of citrullination being in control of cancer progression; however, there is no comprehensive overview of these findings. This article summarizes and critically reviews the influence of citrullination on different aspects of tumor biology, including (i) regulation of apoptosis and differentiation, (ii) promoting EMT and metastasis, and (iii) potential use of citrullinated antigens for immunotherapy. In addition, (iv) the role of citrullination as a cancer biomarker and (v) implication of neutrophil extracellular traps in tumorigenesis are discussed. In summary, current findings testify to the significance of arginine deimination in tumor biology and thus more basic and translational studies are needed to further explore this topic.
Introduction
Citrulline is a noncoding amino acid produced in the body through a posttranslational deimination of peptidyl-arginine (Fig. 1A; ref. 1). This reaction is catalyzed by peptidyl-arginine deiminase (PAD) enzymes, which hydrolyze a guanidino group of the arginine into a urea group, resulting in 1 Da change in molecular mass and converting a positively charged arginine into the electrically neutral citrulline, thereby affecting hydrogen bond formation and protein folding, ultimately resulting in altered hydrophobicity, protein–protein interactions, or even causing denaturation (Fig. 1B; ref. 1–3). Human citrullinome is limited to several hundred proteins, most commonly including vimentin, actin, filaggrin, collagen, fibronectin, keratin, tubulin, and various histones (4, 5).
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.
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.
Exact biological function of citrullination remains obscured; however, arginine deimination is recognized to be central for transcriptional regulation of gene expression (6). Citrullinated histones account for approximately 10% of all histone molecules, emphasizing the significance of this posttranslational modification in many nucleus-associated processes (7). Another known function of citrullination is triggering the formation of neutrophil extracellular traps (NET), a machinery to disable and eliminate bacterial pathogens (8, 9). In neutrophils, PAD4-catalyzed citrullination of histones serves as a starting point for chromatin decondensation and subsequent NET release, enabling to combat infections (Fig. 1B; ref. 10).
Arginine deimination is essential for pathogenesis of several autoimmune diseases, mainly rheumatoid arthritis (RA; ref. 11), and to a lesser extent periodontitis (12), autoimmune encephalomyelitis (13), and systemic lupus erythematosus (SLE; Fig. 1C; ref. 14). Here, deiminated proteins act as neoantigens and inflict the production of autoantibodies, thus boosting a local inflammatory response and exacerbating the severity of disease (1). Importantly, deiminated proteins per se do not seem to serve as an initial inflammatory trigger but uphold and intensify an established inflammatory cycle; hence, citrullination cannot lead to the onset of a disease yet being able to stimulate its progression and aggravation. Antibody responses to citrullinated peptides are used to diagnose RA in clinical setting, whereas multiple studies demonstrated that inhibition of PADs or associated citrullination alleviate RA in animal models (1).
Besides its role in autoimmunity, evidence from multiple translational and clinical studies pointed at the involvement of citrullination in multiple sclerosis, atherosclerosis, thrombosis, and inflammatory bowel disease (Fig. 1C). In these conditions, citrullination acts through quite different mechanisms. In neurological disease, deimination of myelin basic protein reduces its interactions with phospholipids, thereby inhibiting adherence between layers of the myelin sheath and subsequently leading to demyelination (15–17). Pathogenesis of atherosclerosis and thrombosis is aggravated due to NET formation by plaque- or clot-infiltrating neutrophils, providing a feed-forward cycle for disease progression (18). Along similar lines, NETs were detected in crypt abscesses in colonic lesions of patients with ulcerative colitis (19) whereas pharmacologic inhibition of PAD suppressed 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 tumor biology and progression. In this article, I summarize and critically review published studies on this topic.
Brief Overview of PAD Biology and Function in Physiologic Conditions
PADs are a group of five Ca2+-dependent enzymes (PAD1–4 and PAD6), sharing 70% to 95% sequence homology and expressed in a wide range of tissues and organs (Fig. 1D; refs. 22, 23). They contain approximately 650 amino acids and have a molecular weight of 74 kDa (22, 23). Substrate targets of different PADs are determined and partially overlapping (Fig. 1D), but interestingly, not all arginine residues in a protein can be citrullinated. For example, arginine residues in β-turns are much more commonly citrullinated than those in α-helixes, whereas location next to proline or glutamic acid substantially reduces the likelihood of arginine citrullination (24). The most frequent targets for PAD-driven citrullination are keratin, filaggrin, vimentin, actin, histones, collagens, and myelin basic protein (4, 5, 22), all of which having a high arginine content clearly important for their function (Fig. 1D). Notably, free arginine cannot be citrullinated by PADs (22, 25).
In the cellular milieu, all the PADs are localized within the cytosol, but only PAD4 can additionally translocate to the nucleus to citrullinate histones and transcription factors (owing to the presence of a canonical nuclear localization sequence in its structure; refs. 26, 27). Citrullination of the extracellular matrix (ECM) also exists; for example, ECM deposition of PAD4 along with extensive ECM protein citrullination were observed in colorectal cancer liver metastasis with exosomal route of PAD4 delivery being proposed as a mechanism (28). Others, however, speculated that extracellular citrullination can be potentiated by the leak of PADs during cell death (29).
Calcium concentration within the cell is maintained at low levels, thus keeping PADs inactive under physiologic conditions (30). An increase in Ca2+ levels leads to the activation of PADs; however, certain PAD-mediated processes such as gene regulation are described in conditions of physiologic concentrations of calcium, implying the existence of other unknown mechanism of PAD action (27). Apoptosis depends on high cellular concentrations of calcium (31), and protein citrullination is increased in apoptotic cells (30, 32). Cell terminal epidermal differentiation is also dependent on high calcium concentrations accompanied by PAD-induced citrullination of structural proteins such as Keratin (33). The function of PADs as activators and co-activators of transcriptional modulation has also been documented (34, 35), and will be discussed in the following sections of this review. Finally, calcium-independent roles of PADs were reported; in particular, citrullinated proteins are more frequently subjected to degradation potentially due to conformation changes (16, 17), which may additionally implicate PADs in facilitating the turnover of aberrant or redundant proteins. It is important to note that functional difference between PAD family members is still unstudied, yet PAD2 and PAD4 are the most broadly expressed in humans with the latter being involved in several autoimmune diseases.
Prevalence of PADs in Tumor Tissues
Although it is difficult to accurately determine the extent of overall citrullination in tissues and body fluids (36), measuring the corresponding concentration of PAD enzymes appears to be a robust way of estimating the frequency of this posttranslational modification. PAD4 expression levels were increased in various solid tumors relative to their respective normal counterparts (37, 38) as well as overrepresented in the peripheral blood of patients with lung cancer (39). Approximately 40% of cells in malignant lymphomas also expressed PAD4, indicating that expression of this protein is associated with cancer development from all embryological lineages. Benign tumors and nontumor inflamed tissues did not express PAD4 (37), whereas metastasis exhibited much higher PAD4 levels compared with corresponding primary tumors (28), implicating citrullination in the progression from benign neoplasm to invasive malignancy. Taken together, these data indicate that PAD4 is not uncommonly overexpressed in tumors and thus may represent a biomarker or a putative therapeutic target for cancer treatment.
Although tumor-associated PAD2 was overexpressed in biopsies from patients with castration-resistant prostate cancer (CRPC; ref. 40), a downregulation of intertumoral PAD2 expression was identified in a cohort of patient with colorectal cancer specimens in comparison with normal mucosa of healthy control tissues (41).
Because PADs 2 and 4 are the most broadly expressed in humans, other members of this family have not been extensively studied with regards to their abundance in cancer tissues.
Citrullination Affecting Cancer Cell Signaling
PADs and citrullination regulate gene transcription, mainly acting as de-modifying factors to remove the histone (or any other protein) methylation tag and converting the protein to its basal state (42). Because DNA methylation is a key mediator of many physiologic and pathologic conditions (43), replacing the methyl group from arginine serves as powerful regulatory mechanism controlled by PADs. As such, citrullination acts as on/off switch between different modes of transcriptional regulation, being particularly relevant for cancer, a disease strictly dependent on the activation of specific cell signaling programs. As an apt example, after PAD4-driven citrullination of methylated histone H3 Arg17, which is known to regulate estrogen-responsive genes, MCF-7 breast cancer cells displayed a drastic reduction in β-estradiol–induced genes, altering cell phenotype (42). Nonetheless, removal of methylation events by PADs remains largely unstudied with only anecdotical experiments described in literature.
Multiple interactions between PAD4 and p53 were reported, suggesting the importance of PAD-induced citrullination in apoptosis. Expression of a major p53 target gene OKL38 was repressed by a p53-mediated recruitment of PAD4 to the promoter of OKL38 and subsequent removal of histone arginine methylation mark, thus directly modulating apoptosis (44). In particular, inhibition of PADs by their inhibitor Cl-amidine in breast and osteosarcoma cells resulted in OKL38 gene expression, and overexpression of this gene in cell culture led to apoptosis and mitochondria structural changes accompanied by release of cytochrome c (44).
PAD4 was found to bind and subsequently citrullinate the inhibitor of growth 4 (ING4), another tumor suppressor protein known to bind p53 (45). 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 (45).
Interaction of PAD4 with p53 was shown to citrullinate histone H4R3 upon chemotherapy treatment (46), with citrullinated regions being colocalized with decondensed soluble chromatin in apoptotic cells, suggesting the direct involvement of the PAD4–pp53 complex in apoptosis. PAD4−/− mice displayed apoptosis resistance, whereas patients with lung cancer with citrullinated H4R3 expression had smaller tumor size compared with individuals with non-citrullinated H4R3 (46).
In another study (6), histone deacetylase 2 (HDAC2) was determined as a PAD4-interacting protein and they both were shown to bind to p53 and simultaneously associate with the p21 promoter in response to DNA damage in order to regulate gene expression. In addition, 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 (6). Consistent with these reports, overexpression of PAD4 in hematopoietic cancer cell lines increased p53 and p21 expression and directly induced apoptosis (47). Collectively, these studies point at the existence of versatile interactions between p53, proteins belonging to the p53 pathway, and PAD4. Interestingly, such relationship has not been described for other PADs, suggesting that PAD4 has a primary role in apoptosis regulation.
RNA polymerase II (RNAP2) coordinates gene expression by influencing transcription elongation (48). Recently, PAD2 was shown to citrullinate RNAP2 in T47D breast cancer cells, activating transcription of thousands of genes. Inhibition of citrullination by Cl-amidine, PAD2 gene silencing, or mutation of R1810 (site of RNAP2 citrullination) equally reduced cell proliferation through the cell-cycle arrest at the G1 phase (49). Interestingly, other members of the PAD family were unable to citrullinate RNAP2 and induce the above-mentioned effects (49).
Wnt/β-catenin signaling is important for carcinogenesis of certain tumors, and PAD2-mediated citrullination was recently linked to this pathway (50). Antiparasitic drug temozolomide (NTZ) inhibited Wnt/β-catenin signaling through directly targeting PAD2 as demonstrated by co-immunoprecipitation studies. Addition of NTZ resulted in a significant increase of β-catenin citrullination by PAD2 and also substantially extended the half-life of PAD2 protein, suggesting that NTZ enhances stability of PAD2. Further, colony growth assays suggested that PAD2-mediated citrullination of β-catenin could limit the proliferation of colon cancer cells by inhibiting the Wnt pathway. As a result, this elegant study uncovered a hitherto unrecognized mechanism of Wnt signaling inhibition via PAD2. In accord, other studies independently proposed TMZ as a therapeutic target in colon adenocarcinoma, a malignancy characterized by frequent Wnt/β-catenin deregulations (51, 52).
Recent evidence suggested the involvement of citrullination in Androgen signaling in prostate cancer (40). PAD2 was upregulated in patients with CRPC and overexpression of PAD2 by prostate cancer cells induced a CRPC-like phenotype in mouse xenografts, which was mediated by androgen receptor signaling. In particular, PAD2-driven citrullination in the nucleus activated the androgen receptor pathway, whereas PAD2 stabilized the androgen receptor and promoted its translocation to the nucleus. Prostate cancer cells treated with PAD inhibitor Cl-amidine and PAD2-knockdown prostate cancer cells both exhibited a delay in 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 findings. Interestingly, PAD4 expression was shown to be induced by estrogen receptor signaling pathway in MCF-7 breast cancer cells (53), indicating the complexity of the overall picture.
In acute promyelocytic leukemia, mutated malignant cells fail to differentiate into healthy granulocytic cells and undergo uncontrolled self-renewal. A recent elegant study revealed that PAD4 expression is increased during the differentiation of HL-60 leukemia cells induced by the administration of all-trans retinoic acid (ATRA) drug. This cell differentiation depended on the demethylation of the PAD4 promoter (54). Through citrullination of histones, PAD4 stimulated the expression of hematopoietic transcription factors SOX4 and PU.1, which in turn promoted differentiation of HL-60 into granulocytic cells (54). This work highlighted the significance of arginine deimination in cell differentiation, which is supported by a recent extensive study of embryos where PAD4 and citrullination were established as major regulators of pluripotency (55).
Citrullination and Epithelial-to-Mesenchymal Transition
TGFβ signaling has been recognized as critical to cancer cell invasion and metastasis through the activation of the epithelial-to-mesenchymal transition (EMT; ref. 56). To this end, knockdown of PAD4 in breast cancer cells activated TGFβ signaling via upregulation of Smad4, and induced EMT by reducing E-cadherin and promoting vimentin expression, further propelling cell invasiveness (57). These effects were dependent on PAD4-driven citrullination of transcription factor glycogen synthase kinase 3 beta (GSK3β), which resulted in a translocation of GSK3β from the cytoplasm into the nucleus and initiation of multiple gene expression (57). In keeping with these findings, GSK3β was reported to induce EMT (58) and control several transcription factors related to cancer progression (59). Additionally, in a mouse model of hepatic metastases, pharmacological inhibition of PADs abated liver metastatic growth and enhanced the expression of mesenchymal markers, whereas the reverse process, mesenchymal-to-epithelial transition (MET) was suggested to be of importance in initiating metastatic colony formation (28). These studies collectively suggest that PAD4/citrullination axis might be implicated in acquisition of prometastatic phenotype by tumors through regulating EMT.
Consistent with data described above, in human triple-negative breast cancer (TNBC), PAD1 was shown to induce EMT through suppressing ERK1/2 and P38 MAPK signaling through direct citrullination of MEK1 (60). This resulted in disrupting MEK1-mediated phosphorylation of ERK1/2, eventually leading to MMP2 overexpression, eventually triggering EMT.
On the opposite, contradictory findings were provided by Duan and colleagues (61), who reported that upregulation of PAD4 can repress EMT through downregulating the expression of transcription factor Elk1 in lung cancer cell lines (HCC827 and H1650). Interestingly, overexpression of PAD4 in HCC827 and H1650 cells in this study inhibited the resistance of cells to EGFR inhibitor gefitinib through an unknown mechanism (61). To conclude, more research should be done in order to elucidate the importance of citrullination in regulating the EMT plasticity.
NETs Facilitating Cancer
NETs have been detected in several human cancer types (62–65), and some authors raised an interesting possibility of their contribution to cancer development. Chronic inflammation had been suggested to awaken dormant malignant cells, but the mechanism remained elusive (66). Recently, in experimental lung cancer model, inflammation-induced NETs were shown to awaken dormant cancer cells through proteolytic remodeling of laminin, followed by activation of Integrin signaling in tumor cells (67). In particular, NET-derived DNA acted as a proteolysis scaffold by releasing neutrophil elastase and MMP9, which in turn cleaved laminin to generate biologically active epitopes (also known as matrikines) driving cancer cell proliferation (67). These findings indicate that chronic inflammation may provoke cancer recurrence after a long period of dormancy with NETs being a driver of this process. In a different study, the presence of NETs in subcutaneous Lewis lung carcinoma (LLC) grafts increased tumor 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 fewer activated neutrophils. However, priming these neutrophils toward NETosis by the administration of G-CSF promoted NET generation along with an increase in B16 tumor growth (68). This study nonetheless failed to provide a mechanistic explanation of why NETs are required for cancer growth.
Besides promoting tumor cell proliferation and growth, NETs can also be important in metastatic cascade acting as an adhesion substrate for metastasizing cancer cells through Integrin binding (69). Cultured tumor cells exhibited an augmented adhesion to NETs, which was disrupted by pre-incubation with anti-integrin antibodies (69). Furthermore, in a mouse model of sepsis, microvascular NET deposition led to entrapment of circulating lung carcinoma cells within DNA webs, contributing to development of metastases in vivo (70). This process was attenuated by systemic administration of NET inhibitors DNAse or neutrophil elastase inhibitors. The observed NET-mediated trapping of cancer cells within hepatic sinusoids was associated with increased formation of hepatic micrometastases at 48 hours and gross metastatic disease burden at 2 weeks following tumor cell injection (70). Similar to the findings described in ref. 69, the authors demonstrated NETs entrapment of human and murine tumor cells in vitro (70).
Conditions defining NET production in cancers is an intriguing question. Tohme and colleagues (65) demonstrated that isolated mouse neutrophils cultured in media conditioned by colon cancer cells generate NETs in hypoxic conditions characteristic of surgical stress (e.g., cancer-associated hepatectomy). Surgically stressed murine livers were prone to accommodate metastasizing cells and give raise to colonies, whereas administration of NET inhibitors such as DNAse significantly reduced the onset and growth of metastases in surgically stressed but not intact murine livers. Similar results were shown upon the genetic or pharmacologic targeting of PAD4 in mice with experimental liver metastases. These results indicate that surgical stress followed by hypoxia can serve as a requirement for NET generation in the tumor microenvironment. Further mechanistic studies revealed that NETs induced protumorigenic effects through a release of high mobility group box 1 (HMGB1) protein, which in turn activated Toll-like receptor 9 (TLR9) signaling pathway in cancer cells, eventually promoting their proliferation (65). Consistent with these findings, hypoxia induced generation of citrullinated proteins in malignant glioma cells (71). Apart from hypoxia, elevated free fatty acids were also reported to stimulate NET formation concurrently promoting hepatocellular carcinoma growth in mice (72).
Overall, these findings link PAD/citrullination-mediated formation of NETs with tumor growth and metastatic spreading of cancer cells potentially through ECM remodeling, surgical stress, hypoxia, increased fatty acid levels, or through physically binding metastasizing cells to establish colonies. In support of these basic studies results, tumor-infiltrating NETs predicted poor postsurgical survival of pancreatic cancer patients (73), emphasizing the clinical relevance of this phenomenon.
Citrullination Induces Antitumor Immunity
Citrullinated epitopes are presented on MHC-II and stimulate 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 antitumor immunity. In mice bearing subcutaneous tumors, immunization with citrullinated peptides of α-enolase (a key enzyme responsible for Warburg effect in cancers; ref. 75) resulted in a substantial survival advantage in subcutaneous tumor models of melanoma, pancreatic, and lung carcinoma (76). Vaccinated mice demonstrated a stronger CD4+ T-cell response as assessed by IFNγ and granzyme B levels. Interestingly, healthy individuals exhibited a repertoire of CD4+ T cells capable of responding to the citrullinated α-enolase peptide.
In a similar way, Brentville and colleagues (77) vaccinated mice previously challenged with subcutaneous B16 melanoma tumors with citrullinated vimentin peptide, observing an effective anticancer response with 80% of mice surviving after 50 days of the experiment. Administration of blocking anti-CD4 but not anti-CD8 antibodies concurrently with citrullinated vimentin peptide negated the survival advantage, indicating that the observed effect was CD4+ T-cell dependent. This is in concert with findings showing a more pronounced antitumor response to CD4 neo-epitopes in comparison with CD8 neo-epitopes (78).
Collectively, these reports testify to the feasibility of the use of citrullinated peptides as boosters of antitumor immunity and combinations of selected citrullinated peptides in principle may be utilized to target certain cancers.
Citrullination as a Biomarker
Recently, citrullinated histone was proposed as a cancer biomarker in a study where 60 patients with different malignancies of advanced stage exhibited a three-fold increase in the mean concentration of serum citrullinated histone H3 in comparison with 50 healthy individuals or 51 severely ill patients without known cancer (79). These data stipulated that cancer patients have elevated serum levels of citrullinated proteins and that circulating citrullinated histone H3 is not characteristic of general disease burden. In a cohort of cancer patients, invasive tumors had on average more serum citrullinated histone H3 than localized cancers, in agreement with previous reports linking PADs and associated citrullination to metastasis (70). Of note, plasma levels of citrullinated histone H3 correlated with higher levels of cell-free DNA and neutrophil activation in cancer patients, alluding on the involvement of NETs. Strikingly, the authors found a strong correlation between higher plasma levels (>29.8 ng/mL, above the 75th percentile) of citrullinated histone H3 and two-fold increased risk of short-term mortality (79). Because RA is diagnosed by antibody responses to citrullinated proteins (4), it would be of great interest to examine whether clinically used anti-cyclic citrullinated peptide antibodies are successful in detecting malignant neoplasms in prospective or retrospective cohorts.
Synopsis
Posttranslational modifications of human proteome have a major impact on health and disease, expanding the functional diversity of proteins by adding new functional groups. In cancers, many physiologically relevant modifications including citrullination have been infrequently studied thus leaving a potentially important avenue for research. Evidence summarized in this review implicates citrullination in facilitating cancer development through several distinct mechanisms (Table 1; Fig. 2). First, citrullination of histones or transcription factors with subsequent removal of methylation tag dramatically influences fundamental cellular processes such as apoptosis and differentiation. It also affects pathways directly contributing to cancer progression, specifically the Wnt and androgen receptor signaling pathways. It remains unclear, however, what mechanism is in control of such PAD-driven demethylation, whether citrullination of met-Arg residues occurs constantly during the cell cycle or it is entirely context dependent, and if this phenomenon prevails at particular stages of metastatic development or in specific regions of the primary tumor or metastasis.
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 levels of PAD4 in various cancers by IHC (33, 34) and qPCR (35) | Having analyzed 1,673 clinical specimens, the authors found PAD4 to be overexpressed in the carcinomas of the uterus, ovary, colon, bladder, 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. | (37) | |
A total of 162 of 167 individuals with esophageal cancer exhibited high or moderate intratumoral PAD4 levels as evaluated by IHC. Sixteen healthy esophagus specimens showed no signs of PAD4 staining. | (38) | ||
A total of 100 subjects with non–small cell lung cancer demonstrated significantly higher levels of PAD4 in the peripheral blood as compared with 100 healthy blood donors. | (39) | ||
PAD2 | The authors examined the expression of PAD4 in prostate cancer specimens by IHC | The authors found PAD2 to be overexpressed in 27 biopsy specimens from patients with CRPC as compared with 229 specimens from patients with localized prostate cancer. | (40) |
PAD4 | The authors studied the expression of PAD4 in primary colon cancer and associated liver metastasis by immunoblotting and quantitative proteomics | The authors showed that hepatic metastases from five colon cancer liver metastasis specimens exhibited higher PAD4 concentrations compared with five adjacent unaffected livers, five primary colon cancer specimens, or five unaffected colonic mucosas. | (20) |
PAD2 | The authors evaluated expression levels of the PAD4 gene in colon cancer by PCR | Intratumoral PAD2 was downregulated in 98 colorectal cancer patient specimens compared with normal mucosa of 50 healthy control tissues. | (41) |
Citrullination affecting cancer cell signaling PAD4 | |||
The authors investigated citrullination of methylated histone H3 Arg17 in MCF-7 breast cancer cells | Upon methylated histone H3 Arg17 citrullination, there was a drastic reduction in the expression of genes induced by β-estradiol through histone H3 Arg17 methylation. | (42) | |
PAD4 | In MCF-7 breast cancer cells, the authors performed a DNA microarray analysis to identify genes regulated by PAD4 activity in cells treated with Cl-amidine | OKL38 was determined to be a p53 target gene, inducible by DNA damage. Inhibition of PAD4 by Cl-amidine in MCF-7 and U2OS cells resulted in the OKL38 gene expression, and overexpression of the OKL38 gene in cell culture led to apoptosis, mitochondria structure changes, and the delocalization of cytochrome c. Further research showed that OKL38 expression was induced by dynamic p53 and PAD4 binding and histone citrullination. | (44) |
PAD4 | The authors searched for novel protein substrates of PAD4 in vitro | ING4 acted as a substrate for PAD4-induced citrullination. PAD4 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. | (45) |
PAD4 | The authors studied the physiologic role of p53-PAD4 interaction upon the DNA damage using PAD4 deficient mice and clinical cancer tissues | It was found that p53-PAD4 can citrullinate histone H4R3 in adriamycin-treated U2OS cells, and citrullination colocalized with decondensed soluble chromatin in apoptotic cells. PAD4-deficient mice exhibited resistance to apoptosis in response to gamma irradiation–induced DNA damage. Patients with lung cancer with citrullinated H4R3 expression had smaller tumor size compared with individuals with non-citrullinated H4R3. | (46) |
PAD4 | The authors investigated interactions between PAD4, HDAC2, and p53 in U2OS cells | HDAC2 was found to bind to PAD4 and they both were shown to bind to p53 and to associate with the p21 promoter in response to DNA damage 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. | (6) |
PAD4 | The authors examined if inducible overexpression of PAD4 enhances apoptosis in vitro | PAD4 overexpression induced apoptosis in HL-60 and Jurkat cells, and increased the expression of p53, p21, and Bax. | (47) |
PAD2 | The authors sought to explore the antiparasitic drug NTZ in relation to Wnt/β-catenin signaling in multiple models | NTZ administration blocked Wnt/β-catenin signaling through stabilizing PAD2, thus increasing the citrullination and turnover of β-catenin in cancer cells. Colony growth assays showed that PAD2-mediated citrullination of β-catenin limited proliferation of HCT116 and SW480 colon cancer cells by inhibiting the Wnt pathway. | (50) |
PAD2 | The authors explored citrullination of RNAP2 in vitro | In T47D breast cancer cells, PAD2 but not other members of PAD family 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. | (49) |
PAD2 | The authors looked into PAD2 expression in prostate cancer and investigated its interactions with the androgen receptor in vitro and in vivo | PAD2 was found to be overexpressed in patients with CRPC and showed a response to androgen receptor. Levels of PAD4 and citrullinated protein were higher in CRPC tissues than in the localized prostate cancer tissues. Higher levels of PAD2 were associated with elevated levels of 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. | (40) |
PAD4 | The authors were interested in the involvement of PAD4 in differentiation of leukemia cells | PAD4 expression gradually increased during the differentiation of leukemia cells. PAD4 promoter underwent demethylation during HL-60 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. | (54) |
Citrullination and EMT PAD4 | |||
The authors utilized an in vitro gene silencing model to study the effect of PAD4 on TGFβ signaling and EMT | The knockdown of PAD4 in MCF-7 breast cancer cells activated TGFβ signaling via Smad4 and p-Smad2, thus promoting the EMT by reducing E-cadherin and promoting vimentin, ultimately stimulating cell invasiveness. This was dependent on PAD4-mediated citrullination of transcription factor GSK3β, resulting in a translocation of GSK3β from the cytoplasm into the nucleus and triggering gene expression. | (57) | |
PAD4 | The authors treated mice in an experimental liver metastasis experiment with the PAD inhibitor BB-Cl-amidine | Treatment with BB-Cl-amidine reduced metastatic burden in mice. Metastases explanted from untreated animals exhibited high citrullination, upregulated epithelial markers (E-cadherin, cytokeratin 7, 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. | (28) |
PAD1 | The authors explored the molecular mechanisms through which PAD1 affects TNBC invasion using the MDA-MB-231 cell line | PAD1 expression was upregulated in patients with TNBC based on bioinformatics studies. Knockdown or silencing of PAD1 reduced MDA-MB-231 cancer cell proliferation, migration, and invasion in vitro. Further, silencing of PAD1 reduced MMP expression and reversed the EMT in vitro through direct citrullination of MEK1. | (60) |
PAD4 | The authors investigated EMT changes upon the transfection of lung cancer cells HCC827 and H1650 by pCMV-2a/2b-PAD4 transfection or used siRNA to silence PAD4. | Both mRNA and protein levels of Elk1, N-cadherin, and vimentin were substantially downregulated, whereas E-cadherin and α-catenin were upregulated in vitro upon transfection with PAD4 cDNA. In concert, addition of PAD4 siRNA to cells had a reverse impact in comparison with pCMV-2a/2b-PAD4 transfection. In addition, overexpression of PAD4 inhibited the resistance of cells to EGFR inhibitor gefitinib. | (61) |
NETs facilitating cancer PAD4 and NETs | |||
Using multiple models, the authors tested if NETs formed during lung inflammation could induce awakening of dormant metastatic cells. | NETs promoted dormant cancer cell awakening in mice after sustained lung inflammation caused by lipopolysaccharide inhalation or tobacco smoke exposure. NETs also induced dormant cancer cell awakening in vitro in the absence of other host cells. Mechanistically, NET-derived 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 | (67) | |
PAD4 and NETs | Using PAD4−/− mice, the authors addressed the role of the host PAD4/NETs in tumor development. | Unlike NET-free PAD4−/− mice, the presence of NETs in wild-type animals led to an increased tumor growth of LLC grafts. B16 melanoma 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. | (68) |
NETs | The authors performed adhesion assays for different cancer cells using isolated NETs as an adhesion substrate. | Cultured tumor cells U-87 MG, HT-1080, DU 145, PC-3, H1975 exhibited an augmented adhesion to NETs, which was disrupted by pre-incubation with anti-integrin antibodies. There was no effect for A-431 cells in terms of adhesion to NETs. | (69) |
NETs | The authors hypothesized that upon severe infection, NETs are able to trap circulating cancer cells and to promote their early adhesion in distant organ sites | In a mouse model of sepsis (cecal ligation and puncture), the authors observed accumulation of NETs in the liver and lung. Systemic sepsis facilitated hepatic metastasis formation in mice, which was attenuated by administration of DNAse or neutrophil elastase inhibitors. The 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. | (70) |
NETs | The authors hypothesized that upon the surgical stress, NETs can be formed in the liver and may promote the adhesion of circulating tumor cells and enable metastatic growth in mice | Hepatic resection resulted in liver NET formation based on clinical sample assessment. Liver hypoxic microenvironment associated with hepatic resection stimulated NET generation. Surgical stress facilitated liver metastasis in mice, whereas administration of a NET inhibitor DNAse or genetic targeting of PAD4 diminished tumor growth. NETs were shown to induce protumorigenic effects through activation of TLR9 pathway. | (65) |
NETs | The authors investigated a possible link between NETs and nonalcoholic steatohepatitis in vivo, in vitro, and analysing clinical specimens | Analysis of clinical samples showed that nonalcoholic steatohepatitis was characterized by neutrophil infiltration and NET deposition, as well as proinflammatory response. Inhibition of NETs in mice reduced hepatic inflammation and reduced liver cancer progression. In addition, free fatty acids elevated in nonalcoholic steatohepatitis were shown to stimulate NET formation in vitro. | (72) |
NETs | The authors explored the prognostic significance of tumor-infiltrating NETs in pancreatic ductal adenocarcinoma patients | Intratumoral NETs predicted poor postsurgical survival in 317 patients with pancreatic cancer. Multivariate analysis identified NETs as an independent prognostic factor for overall and recurrence-free survival. | (73) |
Citrullination induces antitumor immunity citrullinated α-enolase peptides | |||
The authors questioned whether citrullinated peptides can induce an antitumor immune response in vivo | The authors identified citrullinated α-enolase peptides, which induced a Th1 response in mice as assessed by granzyme B and IFNγ. Vaccination against the citrullinated α-enolase peptide resulted in a strong antitumor effect in melanoma, pancreatic, and lung subcutaneous cancer models in vivo. A T-cell repertoire to citrullinated α-enolase peptides was also identified in humans. | (76) | |
Citrullinated vimentin peptides | The authors questioned if citrullinated peptides can elicit an antitumor immune response in vivo | Immunization of tumor-bearing mice with citrullinated vimentin peptides resulted in effective antitumor responses against B16F1 grafts, which were CD4- but not CD8-mediated and also IFNγ dependent. Immunized mice demonstrated 80% survival on the 40th day after a single dose of citrullinated vimentin peptides injected on day 10 posttumor initiation. | (77) |
Citrullination as a biomarker citrullinated histone H3 | |||
The authors sought to define the levels of circulating citrullinated histone H3 in patients with advanced cancer | Cancer patients had a threefold increase in the median level of citrullinated histone H3 in comparison with healthy individuals and severely ill 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. | (79) |
Subject of the study . | Aim of the study . | Major findings . | Ref. . |
---|---|---|---|
Prevalence of PADs in tumor tissues PAD4 | |||
The authors evaluated expression levels of PAD4 in various cancers by IHC (33, 34) and qPCR (35) | Having analyzed 1,673 clinical specimens, the authors found PAD4 to be overexpressed in the carcinomas of the uterus, ovary, colon, bladder, 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. | (37) | |
A total of 162 of 167 individuals with esophageal cancer exhibited high or moderate intratumoral PAD4 levels as evaluated by IHC. Sixteen healthy esophagus specimens showed no signs of PAD4 staining. | (38) | ||
A total of 100 subjects with non–small cell lung cancer demonstrated significantly higher levels of PAD4 in the peripheral blood as compared with 100 healthy blood donors. | (39) | ||
PAD2 | The authors examined the expression of PAD4 in prostate cancer specimens by IHC | The authors found PAD2 to be overexpressed in 27 biopsy specimens from patients with CRPC as compared with 229 specimens from patients with localized prostate cancer. | (40) |
PAD4 | The authors studied the expression of PAD4 in primary colon cancer and associated liver metastasis by immunoblotting and quantitative proteomics | The authors showed that hepatic metastases from five colon cancer liver metastasis specimens exhibited higher PAD4 concentrations compared with five adjacent unaffected livers, five primary colon cancer specimens, or five unaffected colonic mucosas. | (20) |
PAD2 | The authors evaluated expression levels of the PAD4 gene in colon cancer by PCR | Intratumoral PAD2 was downregulated in 98 colorectal cancer patient specimens compared with normal mucosa of 50 healthy control tissues. | (41) |
Citrullination affecting cancer cell signaling PAD4 | |||
The authors investigated citrullination of methylated histone H3 Arg17 in MCF-7 breast cancer cells | Upon methylated histone H3 Arg17 citrullination, there was a drastic reduction in the expression of genes induced by β-estradiol through histone H3 Arg17 methylation. | (42) | |
PAD4 | In MCF-7 breast cancer cells, the authors performed a DNA microarray analysis to identify genes regulated by PAD4 activity in cells treated with Cl-amidine | OKL38 was determined to be a p53 target gene, inducible by DNA damage. Inhibition of PAD4 by Cl-amidine in MCF-7 and U2OS cells resulted in the OKL38 gene expression, and overexpression of the OKL38 gene in cell culture led to apoptosis, mitochondria structure changes, and the delocalization of cytochrome c. Further research showed that OKL38 expression was induced by dynamic p53 and PAD4 binding and histone citrullination. | (44) |
PAD4 | The authors searched for novel protein substrates of PAD4 in vitro | ING4 acted as a substrate for PAD4-induced citrullination. PAD4 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. | (45) |
PAD4 | The authors studied the physiologic role of p53-PAD4 interaction upon the DNA damage using PAD4 deficient mice and clinical cancer tissues | It was found that p53-PAD4 can citrullinate histone H4R3 in adriamycin-treated U2OS cells, and citrullination colocalized with decondensed soluble chromatin in apoptotic cells. PAD4-deficient mice exhibited resistance to apoptosis in response to gamma irradiation–induced DNA damage. Patients with lung cancer with citrullinated H4R3 expression had smaller tumor size compared with individuals with non-citrullinated H4R3. | (46) |
PAD4 | The authors investigated interactions between PAD4, HDAC2, and p53 in U2OS cells | HDAC2 was found to bind to PAD4 and they both were shown to bind to p53 and to associate with the p21 promoter in response to DNA damage 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. | (6) |
PAD4 | The authors examined if inducible overexpression of PAD4 enhances apoptosis in vitro | PAD4 overexpression induced apoptosis in HL-60 and Jurkat cells, and increased the expression of p53, p21, and Bax. | (47) |
PAD2 | The authors sought to explore the antiparasitic drug NTZ in relation to Wnt/β-catenin signaling in multiple models | NTZ administration blocked Wnt/β-catenin signaling through stabilizing PAD2, thus increasing the citrullination and turnover of β-catenin in cancer cells. Colony growth assays showed that PAD2-mediated citrullination of β-catenin limited proliferation of HCT116 and SW480 colon cancer cells by inhibiting the Wnt pathway. | (50) |
PAD2 | The authors explored citrullination of RNAP2 in vitro | In T47D breast cancer cells, PAD2 but not other members of PAD family 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. | (49) |
PAD2 | The authors looked into PAD2 expression in prostate cancer and investigated its interactions with the androgen receptor in vitro and in vivo | PAD2 was found to be overexpressed in patients with CRPC and showed a response to androgen receptor. Levels of PAD4 and citrullinated protein were higher in CRPC tissues than in the localized prostate cancer tissues. Higher levels of PAD2 were associated with elevated levels of 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. | (40) |
PAD4 | The authors were interested in the involvement of PAD4 in differentiation of leukemia cells | PAD4 expression gradually increased during the differentiation of leukemia cells. PAD4 promoter underwent demethylation during HL-60 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. | (54) |
Citrullination and EMT PAD4 | |||
The authors utilized an in vitro gene silencing model to study the effect of PAD4 on TGFβ signaling and EMT | The knockdown of PAD4 in MCF-7 breast cancer cells activated TGFβ signaling via Smad4 and p-Smad2, thus promoting the EMT by reducing E-cadherin and promoting vimentin, ultimately stimulating cell invasiveness. This was dependent on PAD4-mediated citrullination of transcription factor GSK3β, resulting in a translocation of GSK3β from the cytoplasm into the nucleus and triggering gene expression. | (57) | |
PAD4 | The authors treated mice in an experimental liver metastasis experiment with the PAD inhibitor BB-Cl-amidine | Treatment with BB-Cl-amidine reduced metastatic burden in mice. Metastases explanted from untreated animals exhibited high citrullination, upregulated epithelial markers (E-cadherin, cytokeratin 7, 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. | (28) |
PAD1 | The authors explored the molecular mechanisms through which PAD1 affects TNBC invasion using the MDA-MB-231 cell line | PAD1 expression was upregulated in patients with TNBC based on bioinformatics studies. Knockdown or silencing of PAD1 reduced MDA-MB-231 cancer cell proliferation, migration, and invasion in vitro. Further, silencing of PAD1 reduced MMP expression and reversed the EMT in vitro through direct citrullination of MEK1. | (60) |
PAD4 | The authors investigated EMT changes upon the transfection of lung cancer cells HCC827 and H1650 by pCMV-2a/2b-PAD4 transfection or used siRNA to silence PAD4. | Both mRNA and protein levels of Elk1, N-cadherin, and vimentin were substantially downregulated, whereas E-cadherin and α-catenin were upregulated in vitro upon transfection with PAD4 cDNA. In concert, addition of PAD4 siRNA to cells had a reverse impact in comparison with pCMV-2a/2b-PAD4 transfection. In addition, overexpression of PAD4 inhibited the resistance of cells to EGFR inhibitor gefitinib. | (61) |
NETs facilitating cancer PAD4 and NETs | |||
Using multiple models, the authors tested if NETs formed during lung inflammation could induce awakening of dormant metastatic cells. | NETs promoted dormant cancer cell awakening in mice after sustained lung inflammation caused by lipopolysaccharide inhalation or tobacco smoke exposure. NETs also induced dormant cancer cell awakening in vitro in the absence of other host cells. Mechanistically, NET-derived 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 | (67) | |
PAD4 and NETs | Using PAD4−/− mice, the authors addressed the role of the host PAD4/NETs in tumor development. | Unlike NET-free PAD4−/− mice, the presence of NETs in wild-type animals led to an increased tumor growth of LLC grafts. B16 melanoma 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. | (68) |
NETs | The authors performed adhesion assays for different cancer cells using isolated NETs as an adhesion substrate. | Cultured tumor cells U-87 MG, HT-1080, DU 145, PC-3, H1975 exhibited an augmented adhesion to NETs, which was disrupted by pre-incubation with anti-integrin antibodies. There was no effect for A-431 cells in terms of adhesion to NETs. | (69) |
NETs | The authors hypothesized that upon severe infection, NETs are able to trap circulating cancer cells and to promote their early adhesion in distant organ sites | In a mouse model of sepsis (cecal ligation and puncture), the authors observed accumulation of NETs in the liver and lung. Systemic sepsis facilitated hepatic metastasis formation in mice, which was attenuated by administration of DNAse or neutrophil elastase inhibitors. The 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. | (70) |
NETs | The authors hypothesized that upon the surgical stress, NETs can be formed in the liver and may promote the adhesion of circulating tumor cells and enable metastatic growth in mice | Hepatic resection resulted in liver NET formation based on clinical sample assessment. Liver hypoxic microenvironment associated with hepatic resection stimulated NET generation. Surgical stress facilitated liver metastasis in mice, whereas administration of a NET inhibitor DNAse or genetic targeting of PAD4 diminished tumor growth. NETs were shown to induce protumorigenic effects through activation of TLR9 pathway. | (65) |
NETs | The authors investigated a possible link between NETs and nonalcoholic steatohepatitis in vivo, in vitro, and analysing clinical specimens | Analysis of clinical samples showed that nonalcoholic steatohepatitis was characterized by neutrophil infiltration and NET deposition, as well as proinflammatory response. Inhibition of NETs in mice reduced hepatic inflammation and reduced liver cancer progression. In addition, free fatty acids elevated in nonalcoholic steatohepatitis were shown to stimulate NET formation in vitro. | (72) |
NETs | The authors explored the prognostic significance of tumor-infiltrating NETs in pancreatic ductal adenocarcinoma patients | Intratumoral NETs predicted poor postsurgical survival in 317 patients with pancreatic cancer. Multivariate analysis identified NETs as an independent prognostic factor for overall and recurrence-free survival. | (73) |
Citrullination induces antitumor immunity citrullinated α-enolase peptides | |||
The authors questioned whether citrullinated peptides can induce an antitumor immune response in vivo | The authors identified citrullinated α-enolase peptides, which induced a Th1 response in mice as assessed by granzyme B and IFNγ. Vaccination against the citrullinated α-enolase peptide resulted in a strong antitumor effect in melanoma, pancreatic, and lung subcutaneous cancer models in vivo. A T-cell repertoire to citrullinated α-enolase peptides was also identified in humans. | (76) | |
Citrullinated vimentin peptides | The authors questioned if citrullinated peptides can elicit an antitumor immune response in vivo | Immunization of tumor-bearing mice with citrullinated vimentin peptides resulted in effective antitumor responses against B16F1 grafts, which were CD4- but not CD8-mediated and also IFNγ dependent. Immunized mice demonstrated 80% survival on the 40th day after a single dose of citrullinated vimentin peptides injected on day 10 posttumor initiation. | (77) |
Citrullination as a biomarker citrullinated histone H3 | |||
The authors sought to define the levels of circulating citrullinated histone H3 in patients with advanced cancer | Cancer patients had a threefold increase in the median level of citrullinated histone H3 in comparison with healthy individuals and severely ill 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. | (79) |
Schematic illustrating how PAD-mediated citrullination may affect cell signaling to facilitate cancer progression. PC, prostate cancer.
Schematic illustrating how PAD-mediated citrullination may affect cell signaling to facilitate cancer progression. PC, prostate cancer.
Second, amplification of T-cell immunity by citrullinated antigens, specifically cit-α-enolase and cit-vimentin, may potentially be utilized in cancer immunotherapy. Third, there is some preliminary data suggesting that PADs and associated citrullination are able to enforce EMT thereby contributing to metastasis. Fourth, NETs can also facilitate tumor growth and metastasis seeding via multiple mechanisms, including the entrapment of circulating cancer cells at distant sites and awakening of dormant cells through cleaved laminin peptides (Fig. 3). Finally, serum levels of citrullinated peptides and cell-free DNA characteristic of NETs were proposed as novel biomarkers of solid tumors, which is in line with the fact that many cancers display elevated intratumoral levels of PAD4.
Graphical demonstration of how PAD-driven citrullination of histones promotes NET formation, which in turn facilitate tumor growth and metastasis.
Graphical demonstration of how PAD-driven citrullination of histones promotes NET formation, which in turn facilitate tumor growth and metastasis.
Taken together, these data justify the feasibility of testing PAD inhibitors in preclinical models of cancer. Several irreversible 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 reduced proliferation of cancer cells while not affecting the viability of normal cells (A.E. Yuzhalin; submitted for publication; ref. 81), potentially opening avenues for targeted therapy. Another interesting future direction could be detecting the titers of anti-citrullinated peptide antibodies in cohorts of cancer patients. Having said that, I understand that repertoire of biological functions mediated by citrullination is clearly more diverse than we think, and additional basic research should be further performed to clarify the molecular basis underlying the influence of PADs and associated deimination on tumor progression.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was not financially supported by any funding source.