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Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor

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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.

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Acknowledgments

The authors would like to thank Dr. Joanne Tonkin and Dr. Tommaso Nastasi for kindly providing the IGF1 pro-form specific constructs for the cell transfections.

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Authors and Affiliations

  1. Department of Biomolecular Sciences, Hygiene Unit, University of Urbino Carlo Bo, Via S. Chiara, 27 - 61029, Urbino, PU, Italy

    Mauro De Santi & Giorgio Brandi

  2. Department of Biomolecular Sciences, Exercise and Health Sciences Unit, University of Urbino Carlo Bo, Urbino, PU, Italy

    Giosuè Annibalini, Elena Barbieri, Luciana Vallorani, Serena Contarelli & Vilberto Stocchi

  3. Epidemiology & Prevention Unit, Department of Preventive & Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

    Anna Villarini & Franco Berrino

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  1. Mauro De Santi
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  2. Giosuè Annibalini
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  4. Anna Villarini
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  9. Giorgio Brandi
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Correspondence to Mauro De Santi.

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Funding

This study was supported by the RF-2009-1,532,789 Ministry of Health Project-Italy.

Conflict of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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De Santi, M., Annibalini, G., Barbieri, E. et al. Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor. Cell Oncol. 39, 149–159 (2016). https://doi.org/10.1007/s13402-015-0263-3

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