title: Down-modulation of expression, or dephosphorylation, of IG20/MADD in tumor necrosis factor-related apoptosis-inducing ligand-resistant thyroid cancer cells makes them susceptible to treatment with this ligand.
abstract: BACKGROUND
  The IG20/MADD gene is overexpressed in thyroid cancer tissues and cell lines, and can contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. The ability of the MADD protein to resist TRAIL-induced apoptosis is dependent upon its phosphorylation by Akt. Interestingly, while TRAIL induces a significant reduction in the levels of phospho-Akt (pAkt) and phospho-MADD (pMADD) in TRAIL-sensitive cells, it fails to do so in TRAIL-resistant cells. In this study, we investigated if MADD phosphorylation by Akt was contributing to TRAIL resistance in thyroid cancer cells.
  
  
  METHODS
  We determined the susceptibility of different thyroid cancer cell lines to TRAIL-induced apoptosis by fluorescence-activated cell sorting (FACS) analysis. We tested for various TRAIL resistance factors by FACS analyses or for IG20/MADD expression by quantitative reverse transcription-polymerase chain reaction. We determined the levels of pAkt and pMADD upon TRAIL treatment in thyroid cancer cells by Western blotting. We tested if down-modulation of IG20/MADD gene expression using shRNA or phosphorylation using a dominant negative Akt (DN-Akt) or pretreatment with LY294002, a PI3 kinase inhibitor, could help overcome TRAIL resistance.
  
  
  RESULTS
  BCPAP and TPC1 cells were susceptible, while KTC1 and FTC133 cells were resistant, to TRAIL-induced apoptosis. The differential susceptibility to TRAIL was not related to the levels of expression of death receptors, decoy receptors, or TRAIL. KTC1 and FTC133 cells showed higher levels of IG20/MADD expression relative to BCPAP and TPC1, and were rendered susceptible to TRAIL treatment upon IG20/MADD knockdown. Interestingly, upon TRAIL treatment, the pAkt and pMADD levels were reduced in TRAIL-sensitive BCPAP and TPC1 cells, while they remained unchanged in the resistant KTC1 and FTC133 cells. While expression of a constitutively active Akt in BCPAP and TPC1 cells rendered them resistant to TRAIL, pretreating KTC1 and FTC133 cells with LY294002 rendered them TRAIL-sensitive. Moreover, expression of a DN-Akt in KTC1 and FTC133 cells reduced the levels of pAkt and pMADD and sensitized them to TRAIL-induced apoptosis.
  
  
  CONCLUSIONS
  Our results show that pMADD is an important TRAIL resistance factor in certain thyroid cancer cells and suggest that down-modulation of either IG20/MADD expression or phosphorylation can render TRAIL-resistant thyroid cancer cells sensitive to TRAIL.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
authors.forename: Liang-Cheng
authors.initials: LC
authors.lastname: Li
authors.forename: Shankara
authors.initials: S
authors.lastname: Jayarama
authors.forename: Tania
authors.initials: T
authors.lastname: Pilli
authors.forename: Lixia
authors.initials: L
authors.lastname: Qian
authors.forename: Furio
authors.initials: F
authors.lastname: Pacini
authors.forename: Bellur S
authors.initials: BS
authors.lastname: Prabhakar
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D053218:Receptors, Death Domain / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D013964:Thyroid Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053319:Tumor Necrosis Factor Decoy Receptors / Q000235:genetics
chemical_list: D002867:Chromones; D053418:Death Domain Receptor Signaling Adaptor Proteins; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; C105852:MADD protein, human; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D053218:Receptors, Death Domain; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D053319:Tumor Necrosis Factor Decoy Receptors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051057:Proto-Oncogene Proteins c-akt