title: A novel role for peptidylarginine deiminases in microvesicle release reveals therapeutic potential of PAD inhibition in sensitizing prostate cancer cells to chemotherapy.
abstract: BACKGROUND
  Protein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs) and has been linked to various cancers. Cellular microvesicle (MV) release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression.
  
  
  BACKGROUND
  We have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am). We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment.
  
  
  METHODS
  Prostate cancer cells (PC3) were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation.
  
  
  CONCLUSIONS
  We report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3) to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX) treatment of prostate cancer cells increased the cytotoxic effect of MTX synergistically. Refined PAD inhibitors may form part of a novel combination therapy in cancer treatment.
authors.affiliation: Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London, UK.
authors.forename: Sharad
authors.initials: S
authors.lastname: Kholia
authors.affiliation: Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London, UK.
authors.forename: Samireh
authors.initials: S
authors.lastname: Jorfi
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USA.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Thompson
authors.affiliation: Department of Chemistry, University of North Florida, Jacksonville, FL, USA.
authors.forename: Corey P
authors.initials: CP
authors.lastname: Causey
authors.affiliation: Department of Neurology, University of Alabama at Birmingham, Birmingham, VA, USA.|Birmingham VA Medical Center, Birmingham, AL, USA.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Nicholas
authors.affiliation: Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London, UK; j.inal@londonmet.ac.uk.
authors.forename: Jameel M
authors.initials: JM
authors.lastname: Inal
authors.affiliation: School of Pharmacy, University College London, London, UK; sigrun.lange@ucl.ac.uk.
authors.forename: Sigrun
authors.initials: S
authors.lastname: Lange
keywords: chloramidine; microvesicles; microvesiculation; peptidylarginine deiminases; prostate cancer cells (PC3)