title: Thrombospondin-1 expression in melanoma is blocked by methylation and targeted reversal by 5-Aza-deoxycytidine suppresses angiogenesis.
abstract: BACKGROUND
  Reversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated.
  
  
  RESULTS
  5-Aza-dC reduced DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein, reversed promoter hypermethylation, and restored TSP1 expression in five melanoma cell lines, while having no effect on TSP1 protein levels in normal human melanocytes. In in vivo neovascularization studies, mice were implanted with melanoma cells (A375) either untreated or treated with 5Aza-dC. Vessels at tumor sites were counted by an observer blinded to treatments and the number of tumor vessels was significantly decreased at pretreated tumor sites. This difference occurred before a significant difference in tumor volumes was seen, yet in further studies the average tumor volume in mice treated in vivo with 5-Aza-dC was decreased by 55% compared to untreated controls. Knockdown of TSP1 expression with shRNA enhanced tumor-induced angiogenesis by 68%. Analyses of promoter methylation status of TSP1 in tumors derived from untreated and treated mice identified 67% of tumors from untreated and 17% of tumors from treated mice with partial methylation consistent with the methylation specific PCR analysis of A375 cells. Examination of methylation patterns in the promoter of TSP1 and comparison of aberrantly methylated TSP1 in melanoma with non-malignant nevi identified a significantly higher frequency of promoter methylation in tumor samples from melanoma patients.
  
  
  CONCLUSIONS
  Pharmacological reversal of methylation silenced TSP1 had functional biological consequences in enhancing angiogenesis inhibition and inducing antitumor effects to decrease murine melanoma growth. Angiogenesis inhibition is an additional mechanism by which epigenetic modulators can have antitumor effects.
authors.affiliation: Taussig Cancer Institute, Translational Hematology & Oncology Research, Cleveland Clinic, Cleveland, OH 44195, USA. lindned@ccf.org
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lindner
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.forename: Rebecca
authors.initials: R
authors.lastname: Haney
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Jacobs
authors.forename: John P
authors.initials: JP
authors.lastname: Fruehauf
authors.forename: Ralph
authors.initials: R
authors.lastname: Tuthill
authors.forename: Ernest C
authors.initials: EC
authors.lastname: Borden
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics*; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D008745:Methylation / Q000187:drug effects; D051379:Mice; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C481348:SPZ1 protein, human; D000077209:Decitabine; D001374:Azacitidine