Inhibition of the IGF signalling pathway in MDA-MB-231 triple-negative breast cancer cells

Introduction: Triple-negative breast cancer (TNBC) is characterised by the absence of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) on malignant cells. Insulin-like growth factors (IGFs) stimulate cell proliferation and promote cell survival in TNBC via receptor phosphorylation and activation of adaptor proteins. The aim of this project is to characterise the expression and activation of the IGF signalling pathway in a TNBC cell line, namely MDA-MB-231. Methods: Expression of oestrogen, progesterone and growth hormone receptors and activation of the IGF signalling pathway in MDA-MB-231 cells was analysed by western blotting. The effect of stimulation with IGF1 or inhibition of epidermal growth factor receptor (EGFR)/IGF1R tyrosine kinase activity on proliferation was assessed using an MTS cell proliferation assay. Proliferation was expressed relative to untreated controls, and data was analysed by ANOVA with Tukey’s multiple comparison post hoc test. Results: MDA-MB-231 cells express EGFR and high levels of insulin-like growth factor binding protein 4 (IGFBP4). Moreover, MDA-MB-231 cells express type I IGF1 receptors and proteins in the IGF signalling cascade, namely Erk and Akt. The presence of phosphorylated forms of these proteins suggests activation of the IGF1R signal transduction pathway in MDA-MB-231 cells. Proliferation is increased by IGF1 (E3R), a recombinant IGF1 resistant to binding by IGFBPs. Inhibition of EGFR tyrosine kinase activity or IGF1R tyrosine kinase activity inhibits proliferation of MDA-MB-231 cells. Conclusion: These results suggest that the IGF1 signalling pathway is activated in MDA-MB-231 TNBC cells. Therefore, inhibition of the IGF1R and/or its downstream targets may be of benefit in the treatment of TNBC </p

Recombinant PAPP-A resistant insulin-like growth factor binding protein 4 (dBP4) inhibits angiogenesis and metastasis in a murine model of breast cancer.

BACKGROUND: The Insulin-like growth factor (IGF) pathway plays a role in tumour development and progression. In vivo, IGF1 activity is regulated by the IGF binding proteins (IGFBPs). IGFBP4 inhibits the activity of IGF1 but proteolytic cleavage by pregnancy-associated plasma protein-A (PAPP-A) releases active IGF1. A modified IGFBP4, dBP4, which was resistant to PAPP-A cleavage but retained IGF1 binding capacity, was engineered, expressed in Human Embryonic Kidney (HEK) 293 cells and purified. This study examined the effects of dBP4 on IGF1-induced cell migration, invasion and angiogenesis in vitro. The effect of intra-tumour injections of dBP4 on tumour angiogenesis and metastasis was examined using the 4T1.2luc orthotopic model of breast cancer. METHODS: PAPP-A resistance and IGF binding capacity of dBP4 were characterized by Western blot and surface plasmon resonance, respectively. 4T1.2luc are mouse mammary adenocarcinoma cells transfected with luciferase to allow in vivo imaging. The effect of dBP4 on IGF1-induced Akt activation in 4T1.2luc cells was assessed by Western blot. Cell migration and invasion assays were performed using 4T1.2luc cells. Angiokit™ assays and Matrigel® implants were used to assess the effects of dBP4 on angiogenesis in vitro and in vivo, respectively. An orthotopic breast cancer model - 4T1.2luc cells implanted in the mammary fat pad of BALB/c mice - was used to assess the effect of intra tumour injection of purified dBP4 on tumour angiogenesis and metastasis. Tumour growth and lung metastasis were examined by in vivo imaging and tumour angiogenesis was evaluated by CD31 immunohistochemistry. RESULTS: Our engineered, PAPP-A resistant IGFBP4 (dBP4) retained IGF1 binding capacity and inhibited IGF1 activation of Akt as well as IGF1-induced migration and invasion by 4T1.2 mammary adenocarcinoma cells. dBP4 inhibited IGF1-induced angiogenesis in vitro and in Matrigel implants in vivo. Direct intra-tumour injection of soluble dBP4 reduced angiogenesis in 4T1.2 luc mammary tumours tumour and reduced lung metastasis. CONCLUSION: A PAPP-A resistant IGFBP4, dBP4, inhibits angiogenesis and metastasis in 4T1.2 mammary fat pad tumours. This study highlights the therapeutic potential of dBP4 as an approach to block the tumour-promoting actions of IGF1.</p