title: Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy.
abstract: BACKGROUND
  The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours.
  
  
  METHODS
  Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used.
  
  
  RESULTS
  The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells.
  
  
  CONCLUSIONS
  The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.
authors.affiliation: Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK.
authors.forename: Loreta M
authors.initials: LM
authors.lastname: Rodrigues
authors.forename: Yuen-Li
authors.initials: YL
authors.lastname: Chung
authors.forename: Nada M S
authors.initials: NM
authors.lastname: Al Saffar
authors.forename: Swee Y
authors.initials: SY
authors.lastname: Sharp
authors.forename: Laura E
authors.initials: LE
authors.lastname: Jackson
authors.forename: Udai
authors.initials: U
authors.lastname: Banerji
authors.forename: Marion
authors.initials: M
authors.lastname: Stubbs
authors.forename: Martin O
authors.initials: MO
authors.lastname: Leach
authors.forename: John R
authors.initials: JR
authors.lastname: Griffiths
authors.forename: Paul
authors.initials: P
authors.lastname: Workman
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology* / Q000627:therapeutic use*; D014408:Biomarkers, Tumor / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047029:Lactams, Macrocyclic / Q000494:pharmacology* / Q000627:therapeutic use*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D047368:Tumor Burden / Q000187:drug effects
chemical_list: D016227:Benzoquinones; D014408:Biomarkers, Tumor; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D018719:Erb-b2 Receptor Tyrosine Kinases; C483858:phosphatidylethanolamine; C112765:tanespimycin