Insulin-like growth factors are essential to prevent anoikis in oestrogen-responsive breast cancer cells: importance of the type I IGF receptor and PI3-kinase/Akt pathway

BACKGROUND: Detachment of epithelial cells from the extracellular matrix initiates programmed cell death by a process termed anoikis. Malignant cells must acquire anoikis resistance to leave the primary tumour and metastasise. Multiple signal transduction pathways can activate anoikis and confer anoikis resistance, but these are not understood in breast cancer. METHODS: Models for anoikis of oestrogen-responsive breast cancer cells were established and the protective effects of IGF-1 tested. Cleaved PARP was measured by western transfer and cleaved caspase 3 by flow cytometry. Pathways involved in anoikis and in anoikis resistance were investigated with PI3-kinase, Akt, and MEK1 and MEK2 inhibitors. The importance of the type I IGF receptor was investigated by IGF-concentration dependence, siRNA knockdown and pharmacological inhibition. Association between IGF-1R expression and relapse with distant metastasis was analysed in 1609 patients by log rank test. RESULTS: Unattached breast cancer cells required culture in serum-free medium to induce anoikis. Rapid loss of FAK, Akt and Bad phosphorylation was concurrent with anoiks induction, but ERK1 and ERK2 phosphorylation increased which suggested that anoikis resistance is mediated by the PI3-kinase/Akt rather than the Grb2/Ras/MAP-kinase pathway. IGF-1 conferred anoikis resistance in serum-free medium. IGF-1 activated the PI3-kinase/Akt and Grb2/Ras/MAP-kinase pathways but experiments with PI3-kinase, Akt and MEK1 and MEK2 inhibitors showed that IGF protection is via the PI3-kinase/Akt pathway. The concentration dependence of IGF protection, knockdown experiments with siRNA and pharmacological inhibition with figitumumab, showed that IGF-1 signals through the type I IGF receptor. The crucial role of the type I IGF receptor was demonstrated by induction of anoikis in full serum by figitumumab. High IGF-1R expression was associated with reduced time to relapse with distant metastases in oestrogen receptor-positive patients, especially those with aggressive disease which confirms its relevance in vivo. CONCLUSIONS: Anoikis resistance of oestrogen-responsive breast cancer cells depends upon IGF activation of the type I IGF receptor and PI3-kinase/Akt pathway. Because IGF-dependent evasion of anoikis will facilitate metastasis by malignant breast cancer cells, effective inhibition of IGF signal transduction should be included in combinations of targeted drugs designed to treat metastatic oestrogen receptor-positive breast cancers.