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E002549.Full.Pdf Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2021-002549 on 10 June 2021. Downloaded from Protein citrullination as a source of cancer neoantigens 1 2 3 Hiroyuki Katayama , Makoto Kobayashi, Ehsan Irajizad, 1 1 1 1 Alejandro M Sevillano , Nikul Patel, Xiangying Mao, Leona Rusling, Jody Vykoukal,1 Yining Cai,1 Fuchung Hsiao,1 Chuan- Yih Yu,1 James Long,3 4 5 1 1 Jinsong Liu, Franscisco Esteva, Johannes Fahrmann, Sam Hanash To cite: Katayama H, ABSTRACT modification of proteins via the deamination Kobayashi M, Irajizad E, et al. Background Citrulline post- translational modification of arginine to citrulline. In total, five PADI Protein citrullination as a source of proteins is mediated by protein arginine deiminase of cancer neoantigens. Journal family members are known with sequence (PADI) family members and has been associated with 2 for ImmunoTherapy of Cancer homology ranging from 70% to 95%. To autoimmune diseases. The role of PADI- citrullinome in 2021;9:e002549. doi:10.1136/ date no enzyme has been identified that can jitc-2021-002549 immune response in cancer has not been evaluated. We hypothesized that PADI- mediated citrullinome is a source reverse protein citrullination. The role of of neoantigens in cancer that induces immune response. protein citrullination has been best investi- ► Additional supplemental gated in the context of rheumatoid arthritis material is published online only. Methods Protein expression of PADI family members was To view, please visit the journal evaluated in 196 cancer cell lines by means of indepth (RA), where elevated protein citrullination, online (http:// dx. doi. org/ 10. proteomic profiling. Gene expression was assessed using notably of keratins, filaggrin, vimentin, actin, 1136/ jitc- 2021- 002549). messenger RNA data sets from The Cancer Genome Atlas. histones, nucleophosmin, and nuclear lamin Immunohistochemical analysis of PADI2 and peptidyl- C, has been shown to elicit an autoimmune Accepted 06 April 2021 citrulline was performed using breast cancer tissue response.3 4 Autoimmunity in RA is consid- sections. Citrullinated 12–34-mer peptides in the putative ered to be principally facilitated through Major Histocompatibility Complex-II (MHC-II) binding range Major Histocompatibility Complex (MHC) were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen class II- mediated presentation of citrullinated 5 6 presentation. We further evaluated immunoglobulin- bound peptides that elicit a B cell response. There citrullinome by mass spectrometry using 156 patients with is also currently increased interest in MHC-II breast cancer and 113 cancer-free controls. neoantigens as shaping tumor immunity.7 Results Proteomic and gene expression analyses Comprehensive assessment of the expres- revealed PADI2 to be highly expressed in several cancer sion of PADI family members in cancer has http://jitc.bmj.com/ types including breast cancer. Immunohistochemical been limited. PADI4 has been investigated analysis of 422 breast tumor tissues revealed increased largely with respect to its interactions with © Author(s) (or their expression of PADI2 in ER− tumors (p<0.0001); PADI2 histone H3, ING4, p53, and HDAC2.8–11 employer(s)) 2021. Re- use protein expression was positively correlated (p<0.0001) permitted under CC BY- NC. No with peptidyl-citrulline staining. PADI2 expression exhibited Moreover, the extent to which protein commercial re- use. See rights strong positive correlations with a B cell immune signature citrullination in tumors induces an immune and permissions. Published by response is largely unexplored. Studies aimed on October 2, 2021 by guest. Protected copyright. BMJ. and with MHC- II- bound citrullinated peptides. Increased circulating citrullinated antigen–antibody complexes at interrogating antitumor immunity against 1Clinical Cancer Prevention, The occurred among newly diagnosed breast cancer cases University of Texas MD Anderson citrulline- peptides of vimentin and α-enolase Cancer Center, Houston, Texas, relative to controls (p=0.0012). have demonstrated that these peptides can USA Conclusions An immune response associated with trigger a CD4+ T cell response,12 13 providing 2 Basic Pathology, Fukushima citrullinome is a rich source of neoantigens in breast evidence that protein citrullination in tumors cancer with a potential for diagnostic and therapeutic Medical University, Fukushima, may be immunogenic. Given the antigenicity Japan applications. 3Biostatistics, The University of citrullinated proteins, exploration of PADI of Texas MD Anderson Cancer family members among cancer types and Center, Houston, Texas, USA their impact on citrullination and immune 4 INTRODUCTION Pathology/Laboratory Medicine, response has potential for the development The University of Texas, MD Dysregulated protein citrullination by Anderson Cancer Center, peptidyl arginine deiminases (PADI) has of tumor vaccines with citrullinated antigens Houston, TX, USA been associated with autoimmune diseases, or for identification of citrullinated antigens 5Clinical Development, Cellectis, with recent interest in its relevance to cancer as biomarkers for cancer detection or predic- New York, New York, USA given the occurrence of autoimmunity as a tion of response to immunotherapy.7 14–18 1 2 Correspondence to manifestation of cancer. PADI comprises Herein, we evaluated the protein expres- Professor Sam Hanash; a family of enzymes that, in the presence of sion of PADI family members among 196 shanash@ mdanderson. org calcium ions, catalyze the post-translational cancer cell lines reflective of 12 common Katayama H, et al. J Immunother Cancer 2021;9:e002549. doi:10.1136/jitc-2021-002549 1 Open access cancer types by proteomic profiling as well as by analysis of (EASYnano HPLC System, Thermo Fisher Scientific) J Immunother Cancer: first published as 10.1136/jitc-2021-002549 on 10 June 2021. Downloaded from messenger RNA (mRNA) expression data sets from The coupled online with LTQ Orbitrap ELITE mass spec- Cancer Genome Atlas (TCGA) for 9721 human tumors trometer (Thermo Fisher Scientific). Separations were consisting of 32 different cancer types. Using mass spec- performed using 75 µm inner diameter (id) × 360 µm trometry, we further explored the association of PADI2 od × 25 cm long fused-silica capillary column (Column with the citrullinome of 28 breast cancer cell lines and Technology) slurry packed with 3 µm, 100 A° pore size confirmed the findings of PADI2 expression and citrul- C18 silica- bonded stationary phase. Following injection lination in 422 breast tumors analyzed by immunohisto- of ~500 ng of protein digest onto a C18 trap column chemistry (IHC). We additionally interrogated whether (180 µm id × 20 mm; Waters), peptides were eluted using PADI2- mediated citrullination is associated with a distinct a linear gradient of 0.35% mobile phase B (0.1 formic tumor immunophenotype using TCGA gene expression acid in ACN) per minute for 90 min, then to 95% B for data sets and immunohistochemical analysis of human an additional 10 min, all at a constant flow rate of 300 nL/ breast cancer tumors. We further provide evidence for min. Eluted peptides were analyzed by LTQ Orbitrap circulating autoantibodies against citrullinated tumor- ELITE in a data- dependent acquisition mode. Each full associated proteins in breast cancer. MS scan (400–1800 m/z) was followed by 20 MS/MS scans (Collision- Induced Dissociation (CID) normalized collision energy of 35%). Acquisition of each full mass METHODS spectrum was followed by acquisition of MS/MS spectra Mass spectrometry analyses for the 20 most intense +2, +3, or +4 ions within a duty Breast cancer cell line citrullinome analysis cycle; dynamic exclusion was enabled to minimize redun- Proteomic analysis was performed as previously dant selection of peptides previously selected for MS/MS described.19–22 For indepth citrullinome analysis, 28 breast analysis. Parameters for MS1 were 60,000 for resolution, cancer cell lines (MCF7, MDA- MB-231, SKBR3, HCC1954, 1×106 for automatic gain control target, and 150 ms for HCC1143, BT474, HCC1500, T47D, ZR75-1, HCC1937, maximum injection time. MS/MS was done by CID frag- HCC1599, HCC202, HCC1806, MDA- MB-468, HCC2218, mentation with 3×104 for automatic gain control, 10 ms HCC70, HCC1187, Hs578T, BT549, MCF10A, MCF12A, for maximum injection time, 35 for normalized collision MDA- MB-361, HCC1395, CAMA1, HCC38, MDA- MB- energy, 2.0 m/z for isolation width, 0.25 for activation q- 436, BT20, and MDA- MB-157) were labeled with 13C6 value, and 10 ms for activation time. Lys (#CNLM-2247; Cambridge Isotope Laboratories) in MS/MS spectra were searched against the UniProt RPMI 1640 containing 10% dialyzed Fetal Bovine Serum human proteome database (January 2017) using Sequest (FBS) and 1% penicillin/streptomycin cocktail (Gibco). HT in Proteome Discoverer V.1.4 pipeline. One fixed Stable Isotope Labeling by Amino acid in Cell culture modification of propionamide at Cys (71.037114 Da) (SILAC labeling)23 was performed to discriminate FBS- and three variable modifications, oxidation at Met derived proteins from cell proteins. (15.9949 Da), deamidation at Arg (0.984016 Da), and For proteomic analysis of whole cell lysates, −2×107 SILAC 13C6 at Lys (6.0201 Da), were chosen. The mass http://jitc.bmj.com/ cells were lysed in 1 mL of Phosphate- Buffered Saline error allowed was 10 parts per million (ppm) for parent (PBS) containing octyl- glucoside (1%
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