Brendan C Luey1, Felicity E B May2. 1. Northern Institute for Cancer Research and Newcastle University Institute for Ageing, Department of Pathology, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. 2. Northern Institute for Cancer Research and Newcastle University Institute for Ageing, Department of Pathology, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. F.E.B.May@ncl.ac.uk.
Abstract
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.
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.
Authors: Mark J Mulvihill; Andrew Cooke; Maryland Rosenfeld-Franklin; Elizabeth Buck; Ken Foreman; Darla Landfair; Matthew O'Connor; Caroline Pirritt; Yingchaun Sun; Yan Yao; Lee D Arnold; Neil W Gibson; Qun-Sheng Ji Journal: Future Med Chem Date: 2009-09 Impact factor: 3.808
Authors: Joan M Carboni; Mark Wittman; Zheng Yang; Francis Lee; Ann Greer; Warren Hurlburt; Stephen Hillerman; Carolyn Cao; Glenn H Cantor; Janet Dell-John; Cliff Chen; Lorell Discenza; Krista Menard; Aixin Li; George Trainor; Dolatrai Vyas; Robert Kramer; Ricardo M Attar; Marco M Gottardis Journal: Mol Cancer Ther Date: 2009-12 Impact factor: 6.261
Authors: A M Mercurio; R E Bachelder; J Chung; K L O'Connor; I Rabinovitz; L M Shaw; T Tani Journal: J Mammary Gland Biol Neoplasia Date: 2001-07 Impact factor: 2.673
Authors: Yinghui Zhang; Lynn Wester; Jichao He; Tamar Geiger; Marja Moerkens; Ram Siddappa; Jean A Helmijr; Mieke M Timmermans; Maxime P Look; Caroline H M van Deurzen; John W M Martens; Chantal Pont; Marjo de Graauw; Erik H J Danen; Els M J J Berns; John H N Meerman; Maurice P H M Jansen; Bob van de Water Journal: Oncogene Date: 2018-01-22 Impact factor: 9.867