Glucocorticoid Receptor (NR3C1) Promoter Methylation as a Circulating Breast Cancer Biomarker and its Role in Tamoxifen Sensitivity

Abstract

The glucocorticoid receptor (GR) binds glucocorticoids and transcriptionally regulates genes involved in immune response, cellular proliferation, and apoptosis. Previous work in our lab using MeDIP followed by qPCR found that 15% of assayed breast tumours were methylated at the GR (NR3C1) proximal promoter, and samples with methylation near exon 1B had decreased GR expression compared to unmethylated samples. In this study, a quantitative methylation-specific PCR (qMSP) assay was developed in an attempt to detect NR3C1 promoter methylation in breast cancer patient blood samples. Although circulating NR3C1 promoter methylation was not detectable in our patient cohort, the assay was still used to detect and quantify NR3C1 promoter methylation in the breast tumour samples. Patients with ERα-positive breast tumours with low NR3C1 expression have shown a poorer outcome when treated with tamoxifen compared to tumours with higher NR3C1 expression. Using a doxycycline-inducible lentiviral TRIPZ-shRNAmir in MCF-7 cells directed against endogenous GR, we were able to explore whether low NR3C1 expression modulates cellular response to tamoxifen. When treated with tamoxifen, GR knockdown cells showed increased proliferation compared to normal GR levels, suggesting a decreased sensitivity to tamoxifen. GR knockdown only stimulates proliferation when ERα action is blocked by tamoxifen. Based on these results we propose that NR3C1 promoter methylation detected through qMSP in breast tumours, which leads to low GR expression, may be used as a predictive biomarker for poor outcome in ERα-positive breast tumours treated with tamoxifen.