The anti-estrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF7 breast cancer cells refractory to long-term estrogen deprivation through downregulation of ER and IGF signalling

Long-term culture of MCF-7 wild-type cells in steroid-depleted medium (long-term estrogen deprivation [LTED]) results in hypersensitivity to estradiol (E2) coinciding with elevated levels of ERα, and enhanced type I receptor tyrosine kinase growth factor signalling. In this study we aimed to compare the effects of the pure anti-estrogen ICI 182,780 (ICI) with the competitive anti-estrogen tamoxifen (TAM) on estrogen and IGF signalling in these cells. Wild-type MCF-7 and LTED cells were treated with a seven-log concentration range of E2, TAM or ICI. Effects on cell growth, ERα transactivation, expression of ERα, ERβ, and components of the IGF pathway were measured in the presence of or the absence of insulin. In the presence of insulin, growth of LTED cells was refractory to TAM but was inhibited by ICI and E2. In the absence of insulin, LTED cells showed persistent hypersensitivity to E2, and remained inhibited by ICI but largely unaffected by TAM. Most noteworthy in the absence of insulin, we revealed that E2 doses in excess of 10^-10 M were inhibitory for the LTED cells leading to enhanced apoptosis. ICI but not TAM inhibited ER-mediated gene transcription and resulted in a concomitant dose-dependent reduction in ERα levels, while having no effect on ERβ. As previous studies have suggested that endocrine resistance may result from the non-classical interaction of ERα with AP-1, we transfected the wild-type and LTED cells with an AP-1 reporter construct and monitored activity in response to E2, TAM and ICI. No alteration was detected, suggesting that in this setting transcriptional activity via the ER remained wholly or partially classical via a direct ER/ERE interaction. Analysis of the expression level of proteins within the IGF pathway plus or minus insulin revealed elevated levels of IGF-1 receptor and insulin receptor substrate 2 in LTED versus the wild-type MCF-7 cells, and ICI but not TAM reduced their expression in a dose-dependent fashion. IGF signalling, as well as ERα expression and function, are thus enhanced during LTED. While the resultant cells were resistant to TAM, ICI downregulates ERα, reducing IGF signalling and cell growth. These results support the use of ICI in women with ER-positive breast cancer who have relapsed on an aromatase inhibitor.