PAD4 controls tumor immunity via restraining the MHC class II machinery in macrophages.

Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However, it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here, we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models, accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically, PAD4 citrullinates STAT1 at arginine 121, thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1), and the loss of PAD4 abolishes this interaction, ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus, the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target.

Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody.

BACKGROUND: Peptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer. METHODS: The subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes. RESULTS: PADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated. CONCLUSIONS: PADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.

Transcriptomic analysis of PADI4 target genes during multi-lineage differentiation of embryonic stem cells.

During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that Padi4 expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from Padi4 knock-out (Padi4-KO) mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, Padi4 deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for Padi4 in the specification of the mesodermal lineage and reports the Padi4 associated transcriptome, providing a platform for understanding the physiological functions of Padi4 in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

A common missense variant rs874881 of PADI4 gene and rheumatoid arthritis: Genetic association study and in-silico analysis.

The peptidylarginine-deiminase 4 (PADI4) is involved in the post-translational catalytic conversion of arginine into citrulline. The autoantibodies including anti-citrullinated protein antibodies (ACPAs) produced in response to hypercitrullinated proteins are a hallmark of rheumatoid arthritis (RA) autoimmunity. Therefore, the role of a missense variant rs874881 (Gly112Ala) of PADI4 in RA susceptibility was analyzed, along with in-silico analysis of structural and functional impacts of this substitution. We did a case-control association study and in-silico analysis. For the case-control study, confirmed RA cases and healthy controls were recruited. Genotyping for rs874881 (n = 750) was performed through polymerase chain reaction-restriction fragment length polymorphism. Multivariate logistic regression analysis was employed to determine association. The in-silico analysis was carried out through HOPE, VarMap, MutationAssessor, MutPred2, SIFT, PolyPhen, CADD, REVEL and MetaLR. In the case-control study, the rs874881 exhibited a strong association with increased RA susceptibility (G vs C odds ratio = 3.85, 95 % confidence interval = 2.81-5.27). Interaction analysis revealed significant interaction of genotype with smoking and gender (p < 0.05). Significant results (p < 0.05) were also obtained in stratified analysis by presence/absence of comorbidities and radiographic damage. According to in-silico pathogenicity prediction analysis, this Gly112Ala substitution does not exert a major effect on protein structure and function including its enzymatic activity. We report a significant association of PADI4 rs874881 with overall RA susceptibility. To our knowledge, this is the first study to do the interaction and stratified analyses on the PADI4 rs874881 in RA. Similar detailed studies should also be performed in other populations.

A Screening of Epigenetic Therapeutic Targets for Non-Small Cell Lung Cancer Reveals PADI4 and KDM6B as Promising Candidates.

Despite advances in diagnostic and therapeutic approaches for lung cancer, new therapies targeting metastasis by the specific regulation of cancer genes are needed. In this study, we screened a small library of epigenetic inhibitors in non-small-cell lung cancer (NSCLC) cell lines and evaluated 38 epigenetic targets for their potential role in metastatic NSCLC. The potential candidates were ranked by a streamlined approach using in silico and in vitro experiments based on publicly available databases and evaluated by real-time qPCR target gene expression, cell viability and invasion assays, and transcriptomic analysis. The survival rate of patients with lung adenocarcinoma is inversely correlated with the gene expression of eight epigenetic targets, and a systematic review of the literature confirmed that four of them have already been identified as targets for the treatment of NSCLC. Using nontoxic doses of the remaining inhibitors, KDM6B and PADI4 were identified as potential targets affecting the invasion and migration of metastatic lung cancer cell lines. Transcriptomic analysis of KDM6B and PADI4 treated cells showed altered expression of important genes related to the metastatic process. In conclusion, we showed that KDM6B and PADI4 are promising targets for inhibiting the metastasis of lung adenocarcinoma cancer cells.

The Clinical and Prognostic Significance of Protein Arginine Deiminases 2 and 4 in Colorectal Cancer.

INTRODUCTION: Protein arginine deiminases (PADIs) are a family of enzymes that catalyse the post-translational modification of proteins. Association between PADI expression and clinicopathology, protein expression, and outcome was determined. METHODS: PADI2 and PADI4 expression was assessed immunohistochemically in a cohort of colorectal cancer (CRC) patients. RESULTS: CRC tissues expressed variable levels of PADI2 which was mainly localised in the cytoplasm and correlated with patient survival (p = 0.005); high expression increased survival time from 43.5 to 67.6 months. Expression of cytoplasmic PADI2 correlated with the expression of nuclear ß catenin, PADI4, and alpha-enolase. In contrast, expression of nuclear PADI2 correlated with a decrease in survival (p = 0.010), with high expression decreasing survival from 76.4 to 42.9 months. CRC tissues expressed variable levels of PADI4 in both the nucleus and cytoplasm. Expression of cytoplasmic PADI4 correlated with survival (p = 0.001) with high expression increasing survival time from 48.1 to 71.8 months. Expression of cytoplasmic PADI4 correlated with expression of nuclear ß catenin, alpha-enolase (p ≤ 0.0001, p = 0.002), and the apoptotic related protein, Bcl-2. Expression of nuclear PADI4 also correlated with survival (p = 0.011), with high expression of nuclear PADI4 increasing survival time from 55.4 to 74 months. Expression of nuclear PADI4 correlated with p53, alpha-enolase, and Bcl-2. Multivariate analysis showed that TNM stage, cytoplasmic PADI2, and PADI4 remained independent prognostic factors in CRC. Both PADI2 and PADI4 are good prognostic factors in CRC. CONCLUSION: High expression of cytoplasmic PADI2, PADI4, and nuclear PADI4 were associated with an increase in overall survival.

LINC00324 suppresses apoptosis and autophagy in nasopharyngeal carcinoma through upregulation of PAD4 and activation of the PI3K/AKT signaling pathway.

BACKGROUND: Nasopharyngeal carcinoma (NPC) has high incidence in Southern China and is derived from the mucosal epithelium of the nasopharynx. Accumulating evidence has revealed that peptidyl arginine deiminase 4 (PAD4) exerts carcinogenic effect on certain cancers. We designed this study to probe the specific role that PAD4 plays in NPC and its molecular mechanism. METHODS: PAD4 expression in NPC cells was detected by RT-qPCR analysis. MTT, colony formation, flow cytometry, TUNEL staining, and LC3-II punctuation experiments were done to probe into the biological functions of PAD4 on NPC cellular behaviors in vitro. Subsequently, the upstream regulatory mechanism of PAD4 was investigated by luciferase reporter, RNA pull-down, and RIP assays. The impact of PAD4 on NPC tumor growth in mice was assessed by in vivo xenograft tumor assay. RESULTS: PAD4 was upregulated in NPC cells. PAD4 knockdown suppressed proliferative ability and promoted apoptosis and autophagy in NPC cells. Additionally, PAD4 expression was negatively regulated by microRNA 3164 (miR-3164). LINC00324 positively upregulated PAD4 expression by interacting with miR-3164 and recruiting HuR protein. The LINC00324/miR-3164/PAD4 axis modulated the PI3K/AKT pathway in NPC cells. Moreover, PAD4 upregulation countervailed the influences of LINC00324 deficiency on NPC cell proliferation, apoptosis, and autophagy and on NPC tumor growth in mice. CONCLUSION: LINC00324 promoted NPC malignancy by upregulation of PAD4 to activate the PI3K/AKT pathway.

PADI4 Polymorphisms Confer Risk of Anti-CCP-Positive Rheumatoid Arthritis in Synergy With HLA-DRB1*04 and Smoking.

Peptidylarginine deiminases (PADs) catalyze citrullination, a post-translational modification playing a pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA). The interplay between single nucleotide polymorphisms (SNPs) in the PADI genes and known risk factors for ACPA-positive RA, including smoking, HLA-DR4 and -1, and the PTPN22 R620W polymorphism, was investigated. We typed four PADI2 SNPs, four PADI4 SNPs, and the PTPN22 R620W SNP in 445 Danish RA patients and 533 age-matched healthy controls, as well as in 200 North American RA patients and 100 age- and sex-matched controls. The HLA-DRB1 locus was typed in the Danish cohort. Logistic regression analyses, adjusted for age, sex, smoking status, and PTPN22 R620W, revealed increased risk of anti-CCP-positive RA in carriers of rs11203367(T) (OR: 1.22, p=0.03) and reduced risk in carriers of rs2240335(A) in PADI4 (OR: 0.82, p=0.04). rs74058715(T) in PADI4 conferred reduced risk of anti-CCP-negative RA (OR: 0.38, p=0.003). In HLA-DRB1*04-positive individuals, specifically, the risk of anti-CCP-positive RA was increased by carriage of PADI4 rs1748033(T) (OR: 1.54, p=0.007) and decreased by carriage of PADI4 rs74058715(T) (OR: 0.44, p=0.01), and we observed an interaction between these SNPs and HLA-DRB1*04 (p=0.004 and p=0.008, respectively) Thus, PADI4 polymorphisms associate with ACPA-positive RA, particularly in HLA-DRB1*04-positive individuals, and with ACPA-negative RA independently of HLA-DRB1*04.

The regulation of macrophage polarization by hypoxia-PADI4 coordination in Rheumatoid arthritis.

BACKGROUND: Hypoxia, a common feature of rheumatoid arthritis (RA), induces the over-expression of peptidyl arginine deiminase 4 (PADI4) in fibroblast-like synoviocytes (FLSs) and macrophages. However, the roles of PADI4 and its inducer hypoxia in the regulation of macrophage polarization remain unclear. This study aimed to investigate the role of hypoxia-PADI4 for macrophage polarization in RA patients. METHODS: Synovial tissue (ST) and synovial fluid (SF) were collected from 3 OA patients and 6 RA patients. The distribution of M1 and M2 in ST and cytokines in SF were examined by immunohistochemical analysis and Bio-Plex immunoassays. THP-1 macrophages and BMDM polarization were determined under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions. The effects of PADI4 on macrophages were determined by transfection of adenovirus vector-coated PADI4 (AdPADI4) and the use of PADI4 inhibitor. Finally, the roles of PADI4 in joint synovial lesions on macrophage polarization were investigated in collagen-induced arthritis (CIA) rats. RESULTS: We found increased macrophage polarization of M1 and M2 in the RA ST, compared with OA ST. The ratio of M1/M2 for RA and OA was 1.633 ± 0.1443 and 2.544 ± 0.4429, respectively. The concentration of M1- and M2-type cytokines was higher in RA than that in OA patients. Hypoxia contributed to the increase of the gene and protein expression of M1 and M2 markers. M1- but not M2-type gene expression showed a positive relationship with PADI4 expressionwhile the level of expression of M2-type genes showed no significant difference. The degree of joint swelling and destruction was effectively alleviated, and the number of macrophages especially M1 decreased in CIA rats after down-regulating PADI4 expression. CONCLUSION: Hypoxia is responsible for the co-polarization of M1 and M2. Hypoxia-associated PADI4 is responsible for M1 macrophage activation, implying that the inflammatory environment can be eased by decreasing PADI4 expression and improving the hypoxic environment.

Factors Associated with Promoted Proliferation of Osteosarcoma by Peptidylarginine Deiminase 4.

Osteosarcoma is the most common type of bone malignancy, and the pathogenesis has not been entirely elucidated yet. An important deimination modification enzyme PADI4 (peptidylarginine deiminase 4) has attracted much attention in recent years for its important function in several kinds of human tumors. However, the role of PADI4 on osteosarcoma tumorigenesis remains largely unrevealed. Here, we first assessed the effect of PADI4 on osteosarcoma proliferation by the CCK8 method and colony formation assay. Ectopically expressing PADI4 positively regulates the colony formation capacity of both U2OS and Saos-2 cells. Furthermore, we explored the related mechanism and showed that PADI4 could stimulate Wnt/ß-catenin and MEK/ERK signaling in both U2OS and Saos-2 cells. Then, we detected expression of PADI4 in human tissues of osteosarcoma and revealed that differential expression of PADI4 was associated with tumorigenesis of osteosarcoma. Last, we performed the in vivo experiment in nude mice and results also showed PADI4 could affect the tumor growth. In conclusion, this work revealed that PADI4 could upregulate the proliferation of osteosarcoma, mainly via the Wnt/ß-catenin and MEK/ERK signaling pathway. This study gives us new insight into the regulation mechanism of osteosarcoma proliferation and highlights PADI4 as a promising target for osteosarcoma diagnosis and treatment.

Inhibition of PAD4 enhances radiosensitivity and inhibits aggressive phenotypes of nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a tumor deriving from nasopharyngeal epithelium. Peptidyl-arginine deiminase 4 (PAD4) is a vital mediator of histone citrullination and plays an essential role in regulating disease process. Radiotherapy is an essential method to treat NPC. In this research, we explored the effect of PAD4 on NPC radiosensitivity. METHODS: We enrolled 50 NPC patients, established mice xenograft model, and purchased cell lines for this study. Statistical analysis and a series of experiments including RT-qPCR, clonogenic survival, EdU, Transwell, and wound healing assays were done. RESULTS: Our data manifested that PAD4 (mRNA and protein) presented a high expression in NPC tissues and cells. GSK484, an inhibitor of PAD4, could inhibit activity of PAD4 in NPC cell lines. PAD4 overexpression promoted the radioresistance, survival, migration, and invasion of NPC cells, whereas treatment of GSK484 exerted inhibitory effects on radioresistance and aggressive phenotype of NPC cells. Additionally, GSK484 could attenuate the effect of PAD4 of NPC cell progression. More importantly, we found that GSK484 significantly inhibited tumor size, tumor weight and tumor volume in mice following irradiation. CONCLUSIONS: PAD4 inhibitor GSK484 attenuated the radioresistance and cellular progression in NPC.

Neutrophil extracellular traps mediate joint hyperalgesia induced by immune inflammation.

OBJECTIVE: To evaluate the role of neutrophil extracellular traps (NETs) in the genesis of joint hyperalgesia using an experimental model of arthritis and transpose the findings to clinical investigation. METHODS: C57BL/6 mice were subjected to antigen-induced arthritis (AIA) and treated with Pulmozyme (PLZ) to degrade NETs or Cl-amidine to inhibit NET production. Oedema formation, the histopathological score and mechanical hyperalgesia were evaluated. NETs were injected intra-articularly in wild type (WT), Tlr4-/-, Tlr9-/-, Tnfr1-/- and Il1r-/- mice, and the levels of cytokines and Cox2 expression were quantified. NETs were also quantified from human neutrophils isolated from RA patients and individual controls. RESULTS: AIA mice had increased NET concentration in joints, accompanied by increased Padi4 gene expression in the joint cells. Treatment of AIA mice with a peptidyl arginine deiminase 4 inhibitor or with PLZ inhibited the joint hyperalgesia. Moreover, the injection of NETs into joints of naïve animals generated a dose-dependent reduction of mechanical threshold, an increase of articular oedema, inflammatory cytokine production and cyclooxygenase-2 expression. In mice deficient for Tnfr1, Il1r, Tlr4 and Tlr9, joint hyperalgesia induced by NETs was prevented. Last, we found that neutrophils from RA patients were more likely to release NETs, and the increase in synovial fluid NET concentration correlated with an increase in joint pain. CONCLUSION: The findings indicate that NETs cause hyperalgesia possibly through Toll-like receptor (TLR)-4 and TLR-9. These data support the idea that NETs contribute to articular pain, and this pathway can be an alternative target for the treatment of pain in RA.

Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity.

LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.

Hepatic Surgical Stress Promotes Systemic Immunothrombosis That Results in Distant Organ Injury.

Innate immunity can initiate platelet activation during the development of thrombosis through a process, termed immunothrombosis. Neutrophils form neutrophil extracellular traps (NETs) that have been shown to interact directly with platelets and play pro-coagulant roles in a variety of infectious and sterile inflammatory settings. Hepatic surgical stress initiated by ischemia/reperfusion (I/R) injury has wide systemic consequences on distant organs. However, the mechanisms of this remote injury phenomenon are not well-understood. Here, we sought to determine the role of NETs in causing systemic immunothrombosis and distant organ injury following a local inflammatory insult with liver I/R. Postoperative thromboelastographic revealed that the speed of clot formation (alpha-angle) was significantly increased whereas time to clot formation (R-time) were decreased by in patients undergoing liver resection, indicating a hypercoagulable state after surgery. In mice subjected to liver I/R, circulating platelet activation and platelet-neutrophil aggregates were significantly increased. Injured distant organs such as the lung and kidney displayed NETs and platelet-rich micro-thrombi in the microvasculature following liver I/R. The immune-thrombi and organ damage were dramatically decreased when NETs were inhibited by DNase treatment. Depletion of Tlr4 on platelets limited NET-induced activation of platelets but had no effect on NET formation. Furthermore, platelet-specific TLR4 KO mice had significantly reduced distant organ injury with decreased circulating platelet activation, platelet-neutrophil aggregates following liver I/R in comparison to their control counterparts. These data establish that after an acute local inflammatory process, NET-activated platelets can lead to a systemic pro-coagulant state with resultant remote organ injury by immunothrombosis.

PADI4 modulates the invasion and migration of osteosarcoma cells by down-regulation of epithelial-mesenchymal transition.

Osteosarcoma (OS) is the most common type of primary bone malignancy with high recurrence and metastasis. Peptidylarginine deiminase 4 (PADI4), as an important protein post-translational modification enzyme, has been identified as a potential regulator in the invasion and migration in several types of tumors. The role of PADI4 in osteosarcoma metastasis remains unknown. In this study, we revealed significant positive correlation between PADI4 and pulmonary metastasis of osteosarcoma. Wound-healing and transwell assay indicated that PADI4 induced invasion and migration of osteosarcoma cell in vitro while PADI4 inhibitor has repressive effect. PADI4 mutation with no deimination activity exhibited no significant effect on invasion and migration of osteosarcoma cells. Moreover, we evaluated the effect of PADI4 on expression of the markers of epithelial-mesenchymal transition and results showed that PADI4 promoted EMT while PADI4 inhibitor suppressed EMT in osteosarcoma cells. We also detected the expression of PADI4 and E-Cadherin in the tissues of osteosarcoma patients with or without pulmonary metastasis. Results showed positive relationship between the expression of PADI4 and osteosarcoma metastasis. In contrast, the expression of E-Cadherin exhibited negative correlation with PADI4 and osteosarcoma metastasis. Our research offered a novel link between PADI4 and osteosarcoma metastasis and demonstrated PADI4 as a promising target for treatment of osteosarcoma metastasis.

Stromal Cells Covering Omental Fat-Associated Lymphoid Clusters Trigger Formation of Neutrophil Aggregates to Capture Peritoneal Contaminants.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.

Peptidylarginine Deiminase IV Regulates Breast Cancer Stem Cells via a Novel Tumor Cell-Autonomous Suppressor Role.

Peptidylarginine deiminases (PADI) catalyze posttranslational modification of many target proteins and have been suggested to play a role in carcinogenesis. Citrullination of histones by PADI4 was recently implicated in regulating embryonic stem and hematopoietic progenitor cells. Here, we investigated a possible role for PADI4 in regulating breast cancer stem cells. PADI4 activity limited the number of cancer stem cells (CSC) in multiple breast cancer models in vitro and in vivo. Mechanistically, PADI4 inhibition resulted in a widespread redistribution of histone H3, with increased accumulation around transcriptional start sites. Interestingly, epigenetic effects of PADI4 on the bulk tumor cell population did not explain the CSC phenotype. However, in sorted tumor cell populations, PADI4 downregulated expression of master transcription factors of stemness, NANOG and OCT4, specifically in the cancer stem cell compartment, by reducing the transcriptionally activating H3R17me2a histone mark at those loci; this effect was not seen in the non-stem cells. A gene signature reflecting tumor cell-autonomous PADI4 inhibition was associated with poor outcome in human breast cancer datasets, consistent with a tumor-suppressive role for PADI4 in estrogen receptor-positive tumors. These results contrast with known tumor-promoting effects of PADI4 on the tumor stroma and suggest that the balance between opposing tumor cell-autonomous and stromal effects may determine net outcome. Our findings reveal a novel role for PADI4 as a tumor suppressor in regulating breast cancer stem cells and provide insight into context-specific effects of PADI4 in epigenetic modulation. SIGNIFICANCE: These findings demonstrate a novel activity of the citrullinating enzyme PADI4 in suppressing breast cancer stem cells through epigenetic repression of stemness master transcription factors NANOG and OCT4.

Endogenous PAD4 in Breast Cancer Cells Mediates Cancer Extracellular Chromatin Network Formation and Promotes Lung Metastasis.

Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttranslational modification enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hypercitrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps (NET). NETs were first described as antimicrobial fibers that bind and kill bacteria. However, it is not known whether PAD4 can mediate the release of chromatin DNA into the extracellular space of cancer cells. Here, we report that murine breast cancer 4T1 cells expressing high levels of PADI4 can release cancer extracellular chromatin networks (CECN) in vitro and in vivo. Deletion of Padi4 using CRISPR/Cas9 abolished CECN formation in 4T1 cells. Padi4 deletion from 4T1 cells also reduced the rate of tumor growth in an allograft model, and decreased lung metastasis by 4T1 breast cancers. DNase I treatment, which degrades extracellular DNA including CECNs, also reduced breast to lung metastasis of Padi4 wild-type 4T1 cells in allograft experiments in the Padi4-knockout mice. We further demonstrated that DNase I treatment in this mouse model did not alter circulating tumor cells but decreased metastasis through steps after intravasation. Taken together, our genetic studies show that PAD4 plays a cell autonomous role in cancer metastasis, thus revealing a novel strategy for preventing cancer metastasis by inhibiting cancer cell endogenous PAD4. IMPLICATIONS: This study shows that PADI4 can mediate the formation of CECNs in 4T1 cells, and that endogenous PADI4 plays an essential role in breast cancer lung metastasis. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/5/735/F1.large.jpg.

Ozone-primed neutrophils promote early steps of tumour cell metastasis to lungs by enhancing their NET production.

BACKGROUND: Air pollution, including particulates and gazes such as ozone (O3), is detrimental for patient's health and has repeatedly been correlated to increased morbidity and mortality in industrialised countries. Although studies have described a link between ambient particulate matter and increased lung cancer morbidity, no direct relation has yet been established between O3 exposure and metastatic dissemination to lungs. OBJECTIVES: To outline the mechanisms through which pulmonary O3 exposure modulates metastasis kinetics in an experimental mouse model of O3 exposure. METHODS: Metastatic responses to pulmonary O3 exposure were assessed using a reliable experimental mouse model of concomitant pulmonary O3 exposure and tumour cell injection. Roles of neutrophils in O3-induced lung metastasis were highlighted using blocking anti-Ly6G antibodies; moreover, the implication of neutrophil extracellular traps (NETs) in metastatic processes was evaluated using MRP8cre-Pad4lox/lox mice or by treating mice with DNase I. RESULTS: Pulmonary O3 exposure strongly facilitates the establishment of lung metastasis by (1) Inducing a pulmonary injury and neutrophilic inflammation, (2) Influencing very early steps of metastasis, (3) Priming neutrophils' phenotype to release NETs that favour tumour cell colonisation in lungs. The ability of O3-primed neutrophils to enhance lung colonisation by tumour cells was proven after their adoptive transfer in Balb/c mice unexposed to O3. CONCLUSIONS: Pulmonary neutrophils induced by O3 promote metastatic dissemination to lungs by producing NETs. These findings open new perspectives to improve treatment and prevention strategies in patients affected by metastatic diseases.