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Anti-oestrogen therapy switches off tumour suppressors and proapoptotic genes in breast cancer and reveals a new therapeutic opportunity
Breast Cancer Research volume 10, Article number: P41 (2008)
Previous studies in the Tenovus Centre have demonstrated that the development of antioestrogen resistance in vitro is accompanied by unfavourable changes in the breast cancer phenotype leading to increase tumour cell growth rate. Here evidence is presented to suggest that this is in part due to antihormones causing the epigenetic silencing of oestrogen-induced genes involved in the negative regulation of cell growth. Importantly, we show that reversal of this process using the demethylation agent 5-azacytidine (5AZA) allows oestrogen-induced cell kill by a previously unrecognised mechanism.
The breast cancer cell lines used in this study were MCF7, MCF7-derived tamoxifen-resistant variant (TamR) and TamR sublines that had been withdrawn from tamoxifen (TamRwd) for up to 6 months. Cells were challenged by oestradiol (E2), antihormones and 5AZA. Cell growth responses were assessed by anchorage-dependent growth assays and alterations in expression/activity of oestrogen receptor (ER) and ER-regulated genes were analysed by real-time PCR, western blotting and/or immunocytochemistry.
Compared with the parental MCF7 cells, TamR cells showed a significant upregulated basal rate of growth that was maintained on tamoxifen withdrawal for 6 months. Following the tamoxifen withdrawal, the cells remained ER-positive and showed a slight growth response to E2. In contrast, they showed no growth inhibitory response to tamoxifen. Examination of the methylation status of the promoters of two classically ER-regulated genes switched off in TamR and TamRwd cells, pS2 and progesterone receptor (PR), confirmed their increased methylation and that 5AZA was able to reverse this process, allowing the re-expression of pS2 and PR on E2 treatment. Although pS2 and PR are not thought to play a role in the regulation of cell growth, these data provide proof of principal that gene silencing occurs in TamR cells and that it can be reinstated by 5AZA plus E2. To determine whether tamoxifen was capable of inducing the methylation of ER-regulated genes involved in cell growth, TamRwd cells pretreated with 5AZA were subject to an E2 dose–response challenge. In contrast to TamRwd cells treated with E2, which promoted a growth response, E2 in combination with 5AZA was strongly inhibitory at physiological doses of the steroid (10-9 M), with this action being reversed by tamoxifen. An Affymetrix analysis of the TamR cells has revealed multiple E2-regulated genes that are switched off in the resistant cells whose ontology indicates tumour suppressor/proapoptotic functions.
Our data suggest that antihormone resistance may be associated with the epigenetic silencing of growth inhibitory genes leading to enhanced growth rates. We propose that reinstatement of the expression of such genes using demethylation agents in combination with E2 may provide a previously unrecognised therapeutic opportunity in breast cancer.
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Stone, A., Jones, H., Giles, M. et al. Anti-oestrogen therapy switches off tumour suppressors and proapoptotic genes in breast cancer and reveals a new therapeutic opportunity. Breast Cancer Res 10, P41 (2008). https://doi.org/10.1186/bcr1925
- Progesterone Receptor
- Epigenetic Silence
- Demethylation Agent
- Parental MCF7 Cell