Publications

Abstract

Introduction: Growth?factor pathways are increasingly implicated in tamoxifen resistant cell proliferation; however, targeting of such pathways in oestrogen receptor alpha (ER) positive disease has proven relatively disappointing. In the present study, we have taken the novel approach of using an in?vitro model representative of tamoxifen?withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant?cell phenotype, independent of any agonistic impact of tamoxifen and hence identify potential novel therapeutic approaches for this disease state. Methods: MCF?7?derived tamoxifen?resistant cells routinely maintained in tamoxifen were withdrawn from the drug for 6 months and characterised in terms of ER/EGFR receptor status and anti?hormone and anti?EGFR response. The methylation of ERresponsive gene promoters in tamoxifen?withdrawn cells was confirmed by 5?Azacytidine/oestradiol (5?Aza/E2) challenge with subsequent MethyLight analysis. A microarray approach was used to identify genes that could be associated with the inhibitory effect 5?Aza/E2 co?treatment had on cell proliferation. The methylated status of candidate genes in resistant vs sensitive cells was confirmed using MeDIP material; and as proof?of?concept, one gene candidate, GDF15, was taken forward for functional analysis. Results: Following tamoxifen withdrawal, cells conserved both their resistance to tamoxifen and an increased basal rate of proliferation, despite reduced EGFR expression and reduced sensitivity to gefitinib challenge. Although withdrawn cells retained ER expression, a sub?set of ER?responsive genes were ?silenced? by DNA methylation and their expression could be restored with 5?Aza/E2 co?treatment. Interestingly, an anti?proliferative effect was observed in parallel, and subsequent microarray analysis revealed a cohort of genes that could be associated with this phenomenon. Several gene candidate promoters were more heavily methylated in the resistant vs sensitive cells and restoration of expression was achieved using 5?Aza/E2. Demethylation and subsequent re?expression of one such candidate, GDF15, inhibited cell proliferation. Conclusion: These data provide evidence to support a novel concept that long?term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen?responsive genes whose function is associated with negative cellular proliferation control. Furthermore, reactivation of such genes could provide a potential therapeutic avenue for the management of tamoxifen?resistant breast cancer.