ORIGINAL PAPER Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor Mauro De Santi 1 & Giosuè Annibalini 2 & Elena Barbieri 2 & Anna Villarini 3 & Luciana Vallorani 2 & Serena Contarelli 2 & Franco Berrino 3 & Vilberto Stocchi 2 & Giorgio Brandi 1 Accepted: 3 December 2015 # International Society for Cellular Oncology 2015 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 pathogen- esis of breast cancer have not been established yet. The objec- tive 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 re- spective 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 de- termine 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 ma- ture 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 prolif- eration and IGF1 receptor (IGF1R) phosphorylation, wheras the same antibody was found to only partially inhibit the bio- logical 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 in- duce breast cancer cell proliferation via the IGF1R, indepen- dent 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. Keywords IGF1 pro-forms . Breast cancer . IGF1 receptor 1 Introduction Insulin-like growth factor-1 (IGF1) plays an important role in normal tissue growth and development. In addition, several studies have shown associations between circulating IGF1 levels and the risk to develop breast cancer [1–3]. Since the IGF1 receptor (IGF1R) is over-expressed in about 90 % of the breast cancer cases and since IGF1R levels are higher in breast Electronic supplementary material The online version of this article (doi:10.1007/s13402-015-0263-3) contains supplementary material, which is available to authorized users. * Mauro De Santi mauro.desanti@uniurb.it 1 Department of Biomolecular Sciences, Hygiene Unit, University of Urbino Carlo Bo, Via S. Chiara, 27 - 61029, Urbino, PU, Italy 2 Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy 3 Epidemiology & Prevention Unit, Department of Preventive & Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Cell Oncol. DOI 10.1007/s13402-015-0263-3