A novel role for peptidylarginine deiminases in microvesicle release reveals therapeutic potential of PAD inhibition in sensitizing prostate cancer cells to chemotherapy

Abstract

IntroductionProtein 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.BackgroundWe 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.MethodsProstate 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.ConclusionWe 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.

Peptidylarginine Deiminases as Mediators of Microvesicular Release - Novel Therapeutic Interventions

“…Interestingly, EV shedding from cancer cells also aids increased active drug efflux and thus contributes to their resistance to chemotherapeutic agents [11,52]. In addition, inhibition of microvesiculation has been shown to render cancer cells more susceptible to anticancer drug treatment [7,11] and to reduce the dose of anti-cancer drug docetaxel required to limit tumor growth in vivo [53].…”

“…EVs play physiological roles as mediators of intracellular communication, such as transferring growth factors, microRNAs and enzymes between cells, and play roles in diverse processes such as differentiation, migration and angiogenesis [3][4][5][6][7]. EV release depends on calcium ion influx, which occurs either through pores created by sublytic complement or stimulation of calcium channels such as P2X7 or calcium released by the endoplasmic reticulum through various calcium channels on activated cells [5].…”

“…The cytoskeletal rearrangement is known to be facilitated by the activation of various enzymes, including calpain, gelsolin, scramblase and protein kinase and the simultaneous inhibition of translocase and phosphatases [8]. In addition, recent discoveries on peptidylarginine deiminase (PAD)-mediated deimination of cytoskeletal proteins and histones have elucidated a novel crucial pathway in the facilitation of EV release [7]. As EVs are present in body fluids including blood, urine and cerebrospinal fluid, the identity of circulating EVs may serve as reliable biomarkers of pathophysiological processes [1,2,8,9].…”

“…As EVs are present in body fluids including blood, urine and cerebrospinal fluid, the identity of circulating EVs may serve as reliable biomarkers of pathophysiological processes [1,2,8,9]. EVs are emerging as novel therapeutic targets in treatment of disease as they have been shown to actively contribute to the progression of numerous pathologies including autoimmune diseases [8,10], cancers [7,11,12] and more recently neurodegenerative diseases [9,[13][14][15][16][17][18][19]. Novel discoveries of mechanistic pathways involved, provide potential for the development of new diagnostic assays directed at identifying early stages of disease and response to therapy [1,2].…”

“…Both cellular EV release and PAD-mediated protein deimination are calcium-dependent events that are elevated in a number of human diseases including cancer, autoimmunity and neurodegenerative diseases [10,[24][25][26][27][28][29][30][31]. Addressing the putative interplay between PADs and EV biogenesis, we demonstrated in a recent prostate cancer study that PAD enzyme activation plays a functional role in EV biogenesis through deimination of cytoskeletal actins and nuclear histones [7]. The identification of this novel mechanistic pathway in EV biogenesis has revealed novel synergistic roles for PAD-mediated EV biogenesis in cancer progression.…”