title: Pharmacological targeting of valosin containing protein (VCP) induces DNA damage and selectively kills canine lymphoma cells.
abstract: BACKGROUND
  Valosin containing protein (VCP) is a critical mediator of protein homeostasis and may represent a valuable therapeutic target for several forms of cancer. Overexpression of VCP occurs in many cancers, and often in a manner correlating with malignancy and poor outcome. Here, we analyzed VCP expression in canine lymphoma and assessed its potential as a therapeutic target for this disease.
  
  
  METHODS
  VCP expression in canine lymphomas was evaluated by immunoblotting and immunohistochemistry. The canine lymphoma cell lines CLBL-1, 17-71 and CL-1 were treated with the VCP inhibitor Eeyarestatin 1 (EER-1) at varying concentrations and times and were assessed for viability by trypan blue exclusion, apoptosis by TUNEL and caspase activity assays, and proliferation by propidium iodide incorporation and FACS. The mechanism of EER-1 action was determined by immunoblotting and immunofluorescence analyses of Lys48 ubiquitin and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AFX). TRP53/ATM-dependent signaling pathway activity was assessed by immunoblotting for TRP53 and phospho-TRP53 and real-time RT-PCR measurement of Cdkn1a mRNA.
  
  
  RESULTS
  VCP expression levels in canine B cell lymphomas were found to increase with grade. EER-1 treatment killed canine lymphoma cells preferentially over control peripheral blood mononuclear cells. EER-1 treatment of CLBL-1 cells was found to both induce apoptosis and cell cycle arrest in G1. Unexpectedly, EER-1 did not appear to act either by inducing ER stress or inhibiting the aggresome-autophagy pathway. Rather, a rapid and dramatic increase in γH2AFX expression was noted, indicating that EER-1 may act by promoting DNA damage accumulation. Increased TRP53 phosphorylation and Cdkn1a mRNA levels indicated an activation of the TRP53/ATM DNA damage response pathway in response to EER-1, likely contributing to the induction of apoptosis and cell cycle arrest.
  
  
  CONCLUSIONS
  These results correlate VCP expression with malignancy in canine B cell lymphoma. The selective activity of EER-1 against lymphoma cells suggests that VCP will represent a clinically useful therapeutic target for the treatment of lymphoma. We further suggest a mechanism of EER-1 action centered on the DNA repair response that may be of central importance for the design and characterization of VCP inhibitory compounds for therapeutic use.
authors.affiliation: Département des Sciences Cliniques, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. marie-eve.nadeau@umontreal.ca.
authors.forename: Marie-Ève
authors.initials: MÈ
authors.lastname: Nadeau
authors.affiliation: Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. charlene.m.rico@gmail.com.
authors.forename: Charlène
authors.initials: C
authors.lastname: Rico
authors.affiliation: Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. may_t15@hotmail.com.
authors.forename: Mayra
authors.initials: M
authors.lastname: Tsoi
authors.affiliation: Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. vivancos_melanie@hotmail.fr.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Vivancos
authors.affiliation: Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. sabin.filimon@umontreal.ca.
authors.forename: Sabin
authors.initials: S
authors.lastname: Filimon
authors.affiliation: Département de Pathologie et de Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. marilene.paquet@umontreal.ca.
authors.forename: Marilène
authors.initials: M
authors.lastname: Paquet
authors.affiliation: Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S7C6, Canada. derek.boerboom@umontreal.ca.
authors.forename: Derek
authors.initials: D
authors.lastname: Boerboom
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D004195:Disease Models, Animal; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D015870:Gene Expression; D008198:Lymph Nodes / Q000473:pathology; D008223:Lymphoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000074405:Valosin Containing Protein
chemical_list: D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D016159:Tumor Suppressor Protein p53; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein