Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff, Wales, United Kingdom.
PLoS One. 2012;7(7):e40466. doi: 10.1371/journal.pone.0040466. Epub 2012 Jul 10.
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, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. 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 proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.
在本研究中，我们采用了一种新方法，使用一种代表他莫昔芬停药后临床复发的体外模型，以更深入地了解维持耐药细胞表型的机制，这些机制独立于他莫昔芬的激动作用，从而确定针对这种疾病状态的潜在新治疗方法。在他莫昔芬停药后，即使表皮生长因子受体表达减少，对吉非替尼的敏感性降低，他莫昔芬耐药 MCF-7 细胞在雌激素剥夺环境中仍保留药物耐药性和增殖率的增加。尽管他莫昔芬停药细胞保留 ER 表达，但一组 ER 反应基因，包括 pS2 和孕激素受体（PgR），通过启动子 DNA 甲基化被下调，这一点通过克隆亚硫酸氢盐测序实验得到了证实。用 5-氮杂胞苷（5-Aza）进行启动子去甲基化后，雌二醇（E2）的共添加恢复了这些细胞中的基因表达。此外，5-Aza/E2 共处理在他莫昔芬停药细胞中诱导了显著的抗增殖作用，与单独使用任一药物形成对比。进行了微阵列分析以鉴定受这种共处理特异性上调的基因。鉴定了几个具有抗增殖作用的候选基因，并且它们的启动子在他莫昔芬耐药与敏感细胞中被证实甲基化程度更高。候选基因之一生长分化因子 15（GDF15）被推进进行功能分析。添加 5-Aza/E2 足以使他莫昔芬耐药细胞系中的 GDF15 表达去甲基化和激活，同时，用重组 GDF15 蛋白处理可降低细胞存活率。这些数据提供了证据支持一种新的概念，即长期他莫昔芬暴露诱导一组雌激素反应基因的表观遗传沉默，其功能与负增殖控制有关。此外，使用表观遗传药物重新激活这些基因可能为管理他莫昔芬耐药性乳腺癌提供一种潜在的治疗途径。
