title: 6-Thioguanine inhibition of parathyroid hormone-related protein expression is mediated by GLI2.
abstract: BACKGROUND
  Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GLI2 signaling.
  
  
  METHODS
  Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 μM 6-TG and examined for GLI2 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot.
  
  
  RESULTS
  6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GLI2, the inhibitory effect of 6-TG on PTHrP expression was abolished.
  
  
  CONCLUSIONS
  Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.
authors.affiliation: Department of Veterans Affairs, Tennessee Valley Healthcare System (VISN 9), Nashville, TN, USA.
authors.forename: Rachelle W
authors.initials: RW
authors.lastname: Johnson
authors.forename: Alyssa R
authors.initials: AR
authors.lastname: Merkel
authors.forename: Sabrina
authors.initials: S
authors.lastname: Danilin
authors.forename: Mai P
authors.initials: MP
authors.lastname: Nguyen
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Mundy
authors.forename: Julie A
authors.initials: JA
authors.lastname: Sterling
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D044162:Parathyroid Hormone-Related Protein / Q000037:antagonists & inhibitors* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013866:Thioguanine / Q000494:pharmacology*; D000075746:Zinc Finger Protein Gli2
chemical_list: C082593:GLI2 protein, human; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D044162:Parathyroid Hormone-Related Protein; D012333:RNA, Messenger; D000075746:Zinc Finger Protein Gli2; D013866:Thioguanine