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Endogenous PAD4 in Breast Cancer Cells Mediates Cancer Extracellular

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Endogenous PAD4 in Breast Cancer Cells Mediates Cancer Extracellular Published OnlineFirst March 19, 2020; DOI: 10.1158/1541-7786.MCR-19-0018 MOLECULAR CANCER RESEARCH | NEW HORIZONS IN CANCER BIOLOGY Endogenous PAD4 in Breast Cancer Cells Mediates Cancer Extracellular Chromatin Network Formation and Promotes Lung Metastasis Lai Shi1,2, Huanling Yao3, Zheng Liu3, Ming Xu2,4, Allan Tsung5, and Yanming Wang1,2,3,6 ABSTRACT ◥ Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttransla- Implications: This study shows that PADI4 can mediate the tional modification enzyme that converts protein arginine or mono- formation of CECNs in 4T1 cells, and that endogenous PADI4 methylarginine to citrulline. The PAD4-mediated hypercitrullina- plays an essential role in breast cancer lung metastasis. tion reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps Visual Overview: http://mcr.aacrjournals.org/content/molcanres/ (NET). NETs were first described as antimicrobial fibers that bind 00/00/0000/F1.large.jpg. 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. Introduction opment of distant metastases is a major cause of death from breast cancer. Because of the heterogeneity and lack of well-defined molec- Breast cancer is the most common cancer and the second leading ular targets, patients with metastatic breast cancer have a poor 5-year cause of cancer-related deaths in women in the United States (1). survival rate and finding a method to limit metastasis remains a great Along with the aggressive growth of primary tumor mass, the devel- challenge. Neutrophils are the predominant circulating leukocytes and can promote metastasis through different mechanisms (2–5). Recently, 1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, State College, a novel structure formed by neutrophils, termed neutrophil extra- Pennsylvania. 2The Molecular, Cellular, and Integrative Biosciences Program, cellular traps (NET), was found to play a role in promoting The Pennsylvania State University, State College, Pennsylvania. 3School of metastasis (6–8). NETs were first discovered as a defense mech- Life Sciences, Henan University, Kaifeng, Henan, China. 4Center for Molecular anism utilized by neutrophils to immobilize and kill invading Immunology and Infectious Disease, Department of Veterinary and Biomedical microbes (9). During the past several years, a link between Sciences, The Pennsylvania State University, State College, Pennsylvania. 5 excessive NETs and autoimmunity has been proposed (10). NETs Division of Surgical Oncology, James Cancer Hospital, The Ohio State University fi Wexner Medical Center, Columbus, Ohio. 6School of Medicine, Henan University, also form during brosis, ischemic stroke, preeclampsia, throm- Kaifeng, Henan, China. bosis, cancers, and other autoimmune diseases (11–14). In the context of tumorigenesis, tumors secrete chemokines such as Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). CXCL1 and GCSF to recruit and prime neutrophils to form NETs (6, 15, 16). In turn, NETs facilitate cancer metastasis by Corresponding Author: Yanming Wang, Henan University, Jin Ming Avenue, Kaifeng, Henan 475004, China. Phone: 86-15-6845-36727; E-mail: forming circulating tumor cell (CTC) clusters, enhancing tumor [email protected] cell adhesion to the endothelium, and potentially increasing tumor cell extravasation (7, 17, 18). These studies link NETs to the Mol Cancer Res 2020;XX:XX–XX pathomechanism of metastatic cancer in the context of systemic doi: 10.1158/1541-7786.MCR-19-0018 inflammation and highlight the importance of developing a ther- Ó2020 American Association for Cancer Research. apy for inhibiting NETs. AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 19, 2020; DOI: 10.1158/1541-7786.MCR-19-0018 Shi et al. Peptidyl arginine deiminase 4 (PAD4 or PADI4)-catalyzed histone Animal Care and Use Committee and were conducted in accordance hypercitrullination mediates chromatin decondensation thereby lead- with the NIH Guide for the Care and Use of Laboratory Animals. ing to the formation of NETs (19, 20). PAD4 is a neutrophil-enriched nuclear enzyme that converts arginine and mono-methylarginine on Protein extraction and Western blot analysis histones to citrulline in a calcium-dependent manner (21, 22). Extra- Western blotting using the rabbit a-PAD4 (custom), mouse a-a cellular trap (ET) structures are also formed by other myeloid cells like tubulin (Sigma), rabbit a-histone H3 (Abcam), and rabbit a-H3Cit macrophages and eosinophils, and overexpression of PAD4 is suffi- (Abcam) antibodies was performed essentially as described previous- cient to form NET-like structures in nonhematopoietic cells such as ly (22, 23). The custom a-PAD4 was a rabbit polyclonal antibody made osteosarcoma U2OS cells that normally do not form ETs (20, 23–25). against a human GST-PAD4 fusion protein, and has a similar reac- These findings prompt us to hypothesize that PAD4-mediated hyper- tivity to the conserved human and mouse PAD4 protein (20, 28). citrullination is able to drive ET formation, regardless of cell type. Pathology studies detected significant expression of PAD4 in many Immunostaining and microscopy tumor tissues derived from the colon, duodenum, esophagus, fallopian Antibody staining of cells was performed using standard proto- tube, gall gladder, lung, ovary, parotid, pancreas, prostate, rectum, cols (22). For tissue immunostaining, tumors and lungs were harvested small intestine, stomach, thyroid, and uterus (26, 27). Because PAD4 is from euthanized animals, snap frozen in optimum cutting tempera- also expressed in cancer cells, we investigated whether PAD4 might ture compound cryosectioned, and fixed in zinc fixative (100 mmol/L induce extrusion of chromatin in cancer cells and play a role in cancer Tris-HCl containing 37 mmol/L zinc chloride, 23 mmol/L zinc acetate, progression or metastasis. Here, we report the formation of cancer and 3.2 mmol/L calcium acetate). After fixation, sections were washed extracellular chromatin networks (CECN) both in vitro and in vivo in a with PBST three times, 10 minutes each. Following the third wash, PAD4-dependent manner using the murine breast cancer 4T1 model. tissues were blocked in 2% BSA in PBST for at least 30 minutes at room We found that cancer cell endogenous PAD4 plays a significant role in temperature. Primary antibodies were diluted in PBST supplemented metastasis. In cancer cells without PAD4 expression, tumor metastasis with 2% BSA and 5% normal goat serum. The following primary to the lung was significantly decreased. Our results support that PAD4 antibodies were used: rabbit a-H3Cit (Abcam), rabbit a-H3Cit26 in cancer cells offers a new target for prevention, diagnosis, and (custom), rabbit a-H4Cit3 (Millipore), rabbit a-histone H3 (Abcam), treatment of cancer metastasis. rabbit a-H4Arg3Me (Abcam), mouse a-H4K16Ac (Millipore), rabbit a-HP1a (Active Motif), mouse a-HP1b (Active Motif), mouse a-HP1g (Active Motif), and mouse a-CD45 (eBioscience). The cus- Materials and Methods tom a-H3Cit26 antibody was generated by immunizing rabbits with a Cell culture and transfections synthetic peptide QLATKAA-Cit-KSVPATG corresponding to his- 4T1 and 67NR cells were obtained from Dr. Andrea Mastro (The tone H3 19–33 amino acids with the Cit26 residue. The specificity of Pennsylvania State University, State College, PA). Cell lines were the antibody was confirmed using the same sequence without mod- expanded in our laboratory and stored in liquid nitrogen to ensure ification (histone H3 residues 19–33 QLATKAARKSVPATG). The that cells used for experiments were passaged less than three times. No nuclei were stained by Hoechst 33258 (Sigma). The primary antibodies further genomic authentication was performed but cell lines were were detected by fluorescent dye–conjugated secondary antibodies tested biannually for identity by appearance and growth curve analysis from Jackson ImmunoResearch Laboratories. For terminal deoxynu- and validated to be Mycoplasma free with PCR Mycoplasma Detection cleotidyl transferase–mediated dUTP nick end labeling (TUNEL) Kit (TaKaRa) according to the manufacturer's instruction. To establish staining, sections were stained with Cell Death Detection Kit (Sigma) a stable Padi4 knockout in 4T1, cells were transfected
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