Endocrine Abstracts

Over 70% of breast tumours express oestrogen receptor alpha (ERa). Stimulation with oestradiol results in receptor binding to oestrogen response elements (ERE) in target gene regulatory sequences in association with transcription cofactors, and altered gene expression, resulting in proliferation and tumour growth. Our study aimed to systematically identify genes contributing to ER signalling and oestrogen-dependent proliferation in breast cancer. A genome-wide screen for genes affecting ER signalling was undertaken using a human ER(+) luminal breast cancer cell line (T47DKBLuc) stably expressing a luciferase reporter downstream of three EREs. Stimulation with oestradiol results in a profound increase in luciferase reporter expression, which can be consistently knocked down with shER. Using the Mission shRNA lentiviral library, three shRNA clones were produced for each of >16 000 genes. Following four days of lentiviral infection and oestradiol stimulation, reporter cells were screened for luciferase activity. Primary screening identified 1033 genes (double-hits; 60% cut-off) regulating luciferase reporter expression. We also identified 62 genes (double-hits, 25% cut-off) directly affecting cellular growth. Our hit list includes both known and potentially novel regulators of ER signalling and/or expression. A subset of 614 genes was selected for confirmatory and counter-screening using oestrogen-sensitive (T47DKBLuc, MCF7-ERE-Luc) and insensitive control (T47D-ARE-Luc and MCF7-ARE-Luc; antioxidant response reporter) cells, as well as for assessment of cell proliferation. Using Ingenuity Pathway Analysis (IPA) we confirmed that our screen identified many genes known to be involved in the canonical ER-signalling pathway. In addition, we identified genes involved in other canonical pathways including G-protein coupled receptor signalling, PI3K signalling, and signalling by nuclear receptors glucocorticoid receptor (GR) and androgen receptor (AR), amongst others. This study provides a wealth of data that will give insight into important factors affecting ER biology and proliferation of ER(+) tumours, and reveals potential targets for the treatment of this subclass of breast cancer.