Abstract
We compared mitogenicity and intracellular signalling by human insulin and the AspB10 (X-10) human insulin analogue in MCF-7 human mammary adenocarcinoma cells. By flow analysis of phosphorylated histone H3 or cell cycle distributions, insulin and X-10 were mitogenic at physiologically relevant concentrations (2 nm to 74 pm range), with X-10 being approximately 3-fold more mitogenic than insulin. By western blotting with phospho-specific antibodies, insulin induced phosphorylation of IRS-1, Akt, p70S6K, S6 ribosomal protein, 4E-BP1, FoxO3a, FoxO1, p44/42 MAPK and the EGFR. Blocking with wortmannin, rapamycin and U0126 showed that these signalling events conformed to the canonical PI3K pathway. IRS-1 (Ser302) phosphorylation was abolished by wortmannin and rapamycin, suggesting a feedback from the PI3K pathway on insulin signalling. Compared with equimolar insulin, X-10 caused up to 2-fold higher phosphorylation of all proteins examined in this study. The phosphorylation sites that responded most strongly to insulin were not generally the same as those responding most strongly to X-10. In the PI3K pathway, the most X-10-sensitive protein localized to the translation-regulating arm (p70S6K), with FoxO3a and FoxO1 transcription factors showing a more comparable response to insulin and X-10. Using flow analysis, we confirmed the correlation between insulin-triggered translational activation in G0/G1 (S6 phosphorylation) and S-phase entry by MCF-7 cells. In summary, our findings implicate asymmetrical PI3K pathway activation and specifically stimulation of protein translation in the hypermitogenic effect of insulin analogues such as X-10. It remains to be shown whether these findings are relevant to other human mammary cancer cell types.