title: Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor.
abstract: BACKGROUND
  IGF1 is a key regulator of tissue growth and development and has been implicated in the initiation and progression of various cancers, including breast cancer. Through IGF1 mRNA splicing different precursor pro-peptides, i.e., the IGF1Ea, IGF1Eb and IGF1Ec pro-forms, are formed whose biological roles in the pathogenesis of breast cancer have not been established yet. The objective of this study was to assess the biological activity of the IGF1 pro-forms in human breast cancer-derived cells.
  
  
  METHODS
  The different IGF1 pro-forms were generated through transient transfection of HEK293 cells with the respective vector constructs. The resulting conditioned media were applied in vitro to MCF7, T47D and ZR751 breast cancer-derived cell cultures. The recombinant human IGF1 pro-forms were also tested for their binding affinity to an anti-IGF1 specific antibody by immunoprecipitation. To determine whether the IGF1 pro-forms induce cell proliferation, mature IGF1 was neutralised in HEK293-derived conditioned media.
  
  
  RESULTS
  We found that the IGF1 pro-forms were the only forms that were produced intra-cellularly, whereas both mature IGF1 and the IGF1 pro-forms were detected extra-cellularly. We also found that E peptides can impair the IGF1 pro-form binding affinity for the anti-IGF1 antibody and, thus, hamper an accurate measurement of the IGF1 pro-forms. Additionally, we found that the IGF1 antibody can completely inhibit IGF1-induced breast cancer cell proliferation and IGF1 receptor (IGF1R) phosphorylation, wheras the same antibody was found to only partially inhibit the biological activity of the pro-forms. Moreover, we found that the IGF1 pro-form activities can completely be inhibited by neutralising the IGF1R. Finally, we compared the bioactivity of the IGF1 pro-forms to that of mature IGF1, and found that the IGF1 pro-forms were less capable of phosphorylating the IGF1R in the breast cancer-derived cells tested.
  
  
  CONCLUSIONS
  Our data indicate that IGF1 pro-forms can induce breast cancer cell proliferation via the IGF1R, independent from the mature IGF1 form. These results underline the importance of an accurate assessment of the presence of IGF1 pro-forms within the breast cancer microenvironment.
authors.affiliation: Department of Biomolecular Sciences, Hygiene Unit, University of Urbino Carlo Bo, Via S. Chiara, 27 - 61029, Urbino, PU, Italy. mauro.desanti@uniurb.it.
authors.forename: Mauro
authors.initials: M
authors.lastname: De Santi
authors.affiliation: Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy.
authors.forename: Giosuè
authors.initials: G
authors.lastname: Annibalini
authors.affiliation: Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Barbieri
authors.affiliation: Epidemiology & Prevention Unit, Department of Preventive & Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Villarini
authors.affiliation: Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy.
authors.forename: Luciana
authors.initials: L
authors.lastname: Vallorani
authors.affiliation: Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Contarelli
authors.affiliation: Epidemiology & Prevention Unit, Department of Preventive & Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Berrino
authors.affiliation: Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy.
authors.forename: Vilberto
authors.initials: V
authors.lastname: Stocchi
authors.affiliation: Department of Biomolecular Sciences, Hygiene Unit, University of Urbino Carlo Bo, Via S. Chiara, 27 - 61029, Urbino, PU, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Brandi
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008954:Models, Biological; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011498:Protein Precursors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D014162:Transfection
chemical_list: D003470:Culture Media; D010455:Peptides; D011498:Protein Precursors; C119953:pro-insulin-like growth factor I; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Breast cancer; IGF1 pro-forms; IGF1 receptor