title: Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin).
abstract: BACKGROUND
  Macroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines.
  
  
  METHODS
  Immunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting.
  
  
  RESULTS
  We found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation.
  
  
  CONCLUSIONS
  We show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.
authors.forename: Kamini
authors.initials: K
authors.lastname: Singh
authors.forename: Arishya
authors.initials: A
authors.lastname: Sharma
authors.forename: Maria C
authors.initials: MC
authors.lastname: Mir
authors.forename: Judith A
authors.initials: JA
authors.lastname: Drazba
authors.forename: Warren D
authors.initials: WD
authors.lastname: Heston
authors.forename: Cristina
authors.initials: C
authors.lastname: Magi-Galluzzi
authors.forename: Donna
authors.initials: D
authors.lastname: Hansel
authors.forename: Brian P
authors.initials: BP
authors.lastname: Rubin
authors.forename: Eric A
authors.initials: EA
authors.lastname: Klein
authors.affiliation: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. almasaa@ccf.org.
authors.forename: Alexandru
authors.initials: A
authors.lastname: Almasan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001343:Autophagy; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D000071456:Sequestosome-1 Protein; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D046888:Tissue Array Analysis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D053181:Caspase 8