Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects

Abstract

In MCF-7 breast cancer cells, the insulin-like growth factor 1 receptor (IGF-1R) and the oestrogen receptor (ER) are coexpressed and the two signalling systems are engaged in a crosstalk that results in synergistic growth. However, coupling between the signalling cascades is poorly understood. Oestradiol enhances IGF-1R signalling by inducing the expression of insulin receptor substrate 1 (IRS-1), a substrate of the IGF-1R. Oestradiol induced expression of IRS-1 results in enhanced tyrosine phosphorylation of IRS-1 after IGF-1 stimulation, followed by enhanced mitogen activated protein kinase, phosphoinositide 3' kinase, and Akt activation. Oestradiol can also potentiate the effect of IGF-1 on the expression of cyclin D1 and cyclin E, and on the phosphorylation of the retinoblastoma protein (RB). These effects are greatly diminished in SX13 cells, which exhibit a 50% reduction in IGF-1R expression but few functional IGF-1Rs at the surface. Oestradiol and IGF-1 regulate the expression of two cyclin dependent kinase inhibitors, p21 and p27, differently. Whereas IGF-1 increases p21 expression and reduces p27 expression, oestradiol has no effect on p21. In summary, in MCF-7 cells, oestrogen potentiates the effect of IGF-1 on IGF-1R signalling and its effects on certain cell cycle components.

Figure 1

Schematic representation of the cell cycle. G0, M, G1, S, and G2 refer to the quiescent, mitosis, first gap, DNA synthesis, and second gap phases of the cell cycle, respectively. The two checkpoints (G1–S and G2–M) are shown. The decision to replicate is made at a point during G1 referred to as the G1–S checkpoint or restriction point. Progression up to and through this point in the cell cycle is usually driven by mitogenic growth factors that push the cell into a proliferative mode. RB and pRB represent the unphosphorylated and hyperphosphorylated forms of the retinoblastoma protein.

Figure 2

Abrogation of the synergistic effect of E2 and insulin-like growth factor 1 (IGF-1) on cell growth in SX13 cells. MCF-7 cells were maintained in SFM (serum free medium) in the absence or presence of IGF-1 (1 nM), E2 (10 nM), or a combination of these two mitogens for three days. Cell number was determined indirectly each day using the colorimetric MTT method. Results are expressed as the mean ± SD of percentage of cell number increase as compared with cells maintained in SFM. The results are obtained from three independent experiments using five determinations in each experiment for each condition. *p < 0.05 indicates a significant synergistic effect between IGF-1 and E2.

Figure 3

Effects of E2 and insulin-like growth factor 1 (IGF-1) on the expression and phosphorylation of (A) the IGF-1 receptor β subunit (βIGF-1R), insulin receptor substrate 1 (IRS-1), and Erk1/2 and (B) the phosphoinositide 3¹ (PI3¹) kinase pathways. (A) NEO and SX13 cells were stimulated with IGF-1 (1 nM) for five minutes, E2 (10 nM) for 48 hours, or with both agents. Cells were lysed, proteins separated on sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotted with antibodies to phosphotyrosine (PY20, panels 1 and 3), IGF-1R (panel 2), IRS-1 (panel 4), phospho-Erk1/2 (panel 5), and Erk1 (panel 6, this antibody crossreacts to a lesser extent with Erk2). (B) Cell lysates were immunoprecipitated with αIRS-1 and the PI3′ kinase activity associated with IRS-1 was measured (upper panel). The lower panel represents the protein and serine 473 phosphorylation of Akt in stimulated NEO and SX13 cells (adapted from Dupont and colleagues27).

Figure 4

Expression of surface insulin-like growth factor 1 receptors (IGF-1Rs) in SX13 and NEO cells in response to E2 treatment. Both cell lines were synchronised in G0 phase by serum deprivation and using the anti-oestrogen ICI 182,780 and then maintained in serum free medium in the absence or presence of E2 (10 nM) for 48 hours. Cells were trypsinised and their surface IGF-1R analysed by flow cytometry. In both panels, 1 represents the isotype control (non-specific), 2 represents unstimulated SX13 or NEO cells (starved for 48 hours), and 3 represents SX13 or NEO cells treated with E2 for 48 hours.