Background: CNS tumors are the most frequent solid tumors in pediatric population. The IGF system of ligands and receptors are known to play an important role in both normal and neoplastic growth. Recently, we have shown that in paediatric gliomas, IGF-1R nuclear localization was significantly associated with both high grade tumours and increased risk of death, suggesting that nuclear IGF-1R localization may contribute to an aggressive behaviour of these tumours.
Aim: To characterize the impact of IGF-1R nuclear localization in glioma cells.
Methods: U87Mg glioblastoma cells were transfected with pEGFP-IGF1R plasmid to generate stably transfected cells overexpressing GFP-IGF-1R fusion protein. Corresponding Lys1025- 1100-1120 of the mature IGF-1R were targeted by directed mutagenesis to avoid IGF-1R nuclear translocation. Cells were cultured, starved ON and stimulated 10 min or 8 h with IGF-1. Proliferation assays were carried out during 5 days, with or without IGF-1 stimulation. Wounding assay was analyzed after 18 hours incubation with IGF-1. Protein extracts were obtained from whole lysates or after subcellular fractionation of cultured cells, and processed by western blot using specific antibodies. Gene expression was quantified by rqPCR. All experiments were performed under 50 nM IGF-1 stimulation and 0.5 uM preincubation with OSI906.
Results: We generated stably transfected cells with 5 (B4U87) or 50 (B2U87) times basal expression of IGF-1R. In these cells an increase in pAKT, pERK and pMAPK38 was observed upon 10 min IGF-1 stimulation. Also, after 8 h incubation with IGF-1, nuclear localization of IGF-1R was verified by IF and by western blot of nuclear and cytoplasmic extracts. These effects were blocked by 1 h preincubation with OSI906. Although CyclinD1 levels were slightly increased in B2U87 and B4U87 cells in response to 24 h IGF-1 stimulation, proliferation and apoptosis were not different from parental cells during 5 days of culture. Wounding assay showed an increased motility in both B2U87 and B4U87 compared to parental cells. Moreover, GLUT-1 expression showed a two-fold increase after 24 h rhIGF-1 stimulation that was abrogated by preincubation with OSI906. Cells expressing GFP-IGF-1R1025x-1100x-1120x showed no IGF-1R nuclear localization and a lower increase in GLUT-1 expression upon IGF-1 stimulation.
Conclusion: IGF-1R nuclear localization may contribute to an aggressive phenotype of glioblastoma by increasing motility and metabolism of tumor cells rather than increasing its proliferation.
27 Sep 2018 - 29 Sep 2018