Regulation of the Glucocorticoid Receptor Promoter and Its Prognostic Value in Tamoxifen-Treated ER+ Breast Cancer
Breast Cancer , Glucocorticoid Receptor , Biomarker , Tamoxifen , Estrogen Receptor , Methylation , NR3C1
Most breast cancers are estrogen receptor positive (ER+) and therefore benefit from endocrine-based therapies such as tamoxifen which impair estrogen signaling. However, resistance is a major clinical concern and ER status alone is insufficient to predict treatment response. As a result, additional biomarkers are needed to predict resistance. The glucocorticoid receptor (GR) is an attractive candidate because it functions as a tumor suppressor in ER+ breast cancer and low GR expression has been associated with poor outcome in tamoxifen-treated patients. GR promoter methylation occurs frequently in ER+ tumors and coincides with particularly low GR expression. To investigate GR methylation as a potential biomarker, we developed a targeted multiplex bisulfite sequencing assay to detect promoter methylation in archival tumor samples. With this assay, we evaluated the prognostic and predictive value of GR methylation in ER+ patients from the CCTG MA.12 tamoxifen clinical trial. Region-specific GR promoter methylation was an independent marker of poor prognosis and identified a subset of patients with especially poor survival, particularly in the absence of tamoxifen treatment. This provides a foundation for the continued development of GR methylation as a prognostic marker in ER+ breast cancer. Given the tumor suppressive role of GR in ER+ breast cancer, we examined the regulation of GR expression in the context of tamoxifen treatment. GR mRNA levels increased with tamoxifen in ER+ but not ER− breast cancer cells, suggesting ER is involved in regulating GR expression. The GR promoter is particularly complex with multiple first exons. By adapting 5’RACE for next-generation sequencing, we established that increased GR expression was mediated through the same exons in the proximal promoter in tamoxifen-treated ER+ cells. We then carried out a functional analysis of this promoter region in ER− cells transiently expressing ER or naturally-occurring ER mutants (Y537S or D538G). Our findings suggest tamoxifen upregulates GR through an ER-dependent non-classical signaling mechanism with differing characteristics depending on the ER mutation. Since GR has an established antiproliferative effect on ER+ breast cancer cells, tamoxifen-induced GR upregulation could be an important factor influencing treatment response. This work may have important clinical implications for tamoxifen-treated patients, including those with acquired ER mutations.