Comparison of intracellular signalling by insulin and the hypermitogenic AspB10 analogue in MCF‐7 breast adenocarcinoma cells

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.

Insulin Receptor Phosphorylation by Endogenous Insulin or the Insulin Analog AspB10 Promotes Mammary Tumor Growth Independent of the IGF-I Receptor

“…Differences between in vitro and in vivo potency of AspB10 have been previously described (35) and highlight the need for caution when interpreting the results of in vitro studies. In vivo studies have not previously demonstrated whether AspB10 exerts its mitogenic effects through the IR or IGF-IR (21,25). Understanding the mechanism through which AspB10 promotes tumor growth is important for the development of new insulin analogs to ensure they are not mitogenic.…”

Insulin X10 revisited: a super-mitogenic insulin analogue

“…An asymmetrical preferential phosphatidylinositol 3-kinase (PI3K) pathway activation and, specifically, stimulation of protein translation, was seen after stimulation with insulin X10 [29]. Other studies have also found an important role of the PI3K pathway [25], whereas yet more results seem to favour a more pronounced stimulation of the extracellular signal-regulated kinase (ERK) pathway [30].…”

Role of Insulin in the Type 2 Diabetes Therapy: Past, Present and Future

“…The possibility that insulin analogues have a different effect on the incidence of cancer than human insulin, has been a reason for concern since the 1990s, when the clinical development of the rapid-acting analogue AspB10 had to be terminated because of the risk for malignancies - an effect attributed to a lower dissociation rate from the insulin receptor and/or to a higher affinity for the IGF-1 receptor (23). Glargine also has a greater affinity for the IGF-1 receptor than human insulin, producing a greater proliferative effect in vitro (24).…”