Identification of genes associated with the maintenance of the tamoxifen resistant breast cancer cell phenotype following tamoxifen withdrawal

Despite the benefit tamoxifen has provided for millions of breast cancer patients worldwide, almost all patients with metastatic disease and as many as 40 of patients receiving adjuvant tamoxifen treatment will acquire resistance to the drugs inhibitory effect on breast cancer cell growth. Previous studies in the Tenovus Centre have demonstrated that the development of anti-oestrogen resistance in vitro is associated with aberrant growth factor signaling which facilitates a more aggressive cell phenotype. The aim of the present study was to determine whether the undesirable characteristics of tamoxifen resistant cells were maintained following withdrawal from the drug. Interestingly, the accelerated rate at which resistant cells proliferated was sustained following a 6 month withdrawal period despite decreased expression of epidermal growth- factor receptor and reduced sensitivity to gefitinib. Following the assessment of long-term tamoxifen exposure on classically regulated oestrogen gene targets progesterone receptor and trefoil factor 1, it was apparent that the genes were no longer inducible by oestradiol following the acquisition of resistance. In contrast, when cells were co-treated with a demethylation agent in combination with oestradiol, genes were once again responsive to oestrogen stimulation, providing proof of principle that long-term tamoxifen exposure can silence oestrogen regulated gene expression through promoter hyper- methylation. Importantly, this combination treatment was shown to significantly reduce cell growth, inferring that a proportion of the genes that were reactivated by this treatment were associated with a tumour suppressive function. Using microarray technology, methylight analysis and polymerase chain reaction validation, several genes with tumour-suppressive ontology were identified as being silenced by promoter hypermethylation in tamoxifen-1 withdrawn tamoxifen-resistant cells, including p53 gene target, prostate differentiation factor, and inhibitor of Ras signaling, Ras protein activator-like 1. It is therefore proposed that anti-hormone induced epigenetic modification of tumour-suppressor genes, alongside aberrant growth factor signaling, can promote resistant cell survival and progression