title: Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression in multiple myeloma plasma cells.
abstract: BACKGROUND
  Multiple myeloma is an incurable malignancy of bone marrow plasma cells. Progression of multiple myeloma is accompanied by an increase in bone marrow angiogenesis. Studies from our laboratory suggest a role for the CXCL12 chemokine in this process, with circulating levels of CXCL12 correlating with bone marrow angiogenesis in patients with multiple myeloma. While the mechanisms responsible for aberrant plasma cell expression of CXCL12 remain to be determined, studies in other systems suggest a role for hypoxia and hypoxia-inducible transcription factors.
  
  
  METHODS
  The expression of hypoxia-inducible factor protein was examined in patients' bone marrow biopsy specimens using immunohistochemistry. The hypoxic regulation of CXCL12 was examined in multiple myeloma plasma cell lines using polymerase chain reaction and western blotting. The role of hypoxia-inducible factors-1 and -2 in the regulation of CXCL12 expression was examined using over-expression and short hairpin RNA knockdown constructs, electrophoretic mobility shift assays and chromatin immunoprecipitation. The contribution of CXCL12 to hypoxia-induced angiogenesis was examined in vivo using a subcutaneous murine model of neovascularization.
  
  
  RESULTS
  Strong hypoxia-inducible factor-2 protein expression was detected in CD138(+) multiple myeloma plasma cells in patients' biopsy specimens. Prolonged exposure to hypoxia strongly up-regulated CXCL12 expression in multiple myeloma plasma cells and hypoxia-inducible factor-2 was found to play a key role in this response. Promoter analyses revealed increased hypoxia-inducible factor-2 binding to the CXCL12 promoter under hypoxic conditions. Over-expression of hypoxia-inducible factor in multiple myeloma plasma cells strongly induced in vivo angiogenesis, and administration of a CXCL12 antagonist decreased hypoxia-inducible factor-induced angiogenesis.
  
  
  CONCLUSIONS
  Hypoxia-inducible factor-2 is a newly identified regulator of CXCL12 expression in multiple myeloma plasma cells and a major contributor to multiple myeloma plasma cell-induced angiogenesis. Targeting the hypoxic niche, and more specifically hypoxia-inducible factor-2, may represent a viable strategy to inhibit angiogenesis in multiple myeloma and progression of this disease.
authors.affiliation: Myeloma Research Program, Division of Haematology, Centre for Cancer Biology-SA Pathology and University of Adelaide, Australia.
authors.forename: Sally K
authors.initials: SK
authors.lastname: Martin
authors.forename: Peter
authors.initials: P
authors.lastname: Diamond
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Williams
authors.forename: Luen Bik
authors.initials: LB
authors.lastname: To
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Peet
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Fujii
authors.forename: Stan
authors.initials: S
authors.lastname: Gronthos
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Harris
authors.forename: Andrew C W
authors.initials: AC
authors.lastname: Zannettino
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000502:physiology*; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000096:biosynthesis* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010950:Plasma Cells / Q000378:metabolism / Q000473:pathology*; D023041:Xenograft Model Antitumor Assays; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D054377:Chemokine CXCL12; D000099162:Endothelial PAS Domain-Containing Protein 1; C515255:CXCL12 protein, human