Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

Abstract

BackgroundSignaling by estrogen-receptor alpha (ER) plays a major role in breast cancer initiation Investigations of the mechanism of DNA damage mediated by ER signaling are carried out in breast cancer cell lines due to the lack of ER+ normal human breast epithelial cells lines (HBEC). Defining the mechanisms by which ER induces DNA damage and initiates tumorigenesis requires normal HBECs that express ER, demonstrate estrogenic responses, and are amenable to long term propagation in culture. MethodsWe utilized lentiviral expression of an inducible ER construct to generate four HBEC lines (HBEC-ESR1). We studied these cells for ER-dependent responses using a luciferase reporter, endogenous gene expression and proliferation assays. RNA-Seq was performed to characterize the ER-mediated transcriptomic patterns in the four HBEC lines. ER mediated DNA double strand breaks (DSBs) were analyzed using {gamma}H2AX immunofluorescence. ResultsExpression and functional activation of ER were observed in all HBEC-ESR1 lines, whereas proliferation in response to 17{beta}-estradiol (E2) was observed in 3 of the cell lines. Proliferative responses were due to intrinsic signaling within the HBECs as conditioned media from the cells failed to cause proliferation. A total of 682 genes were differentially expressed at 24h following treatment with 10nM E2 with 43% of these genes were also observed in ER+ breast cancer cell lines (MCF7 or T47D). Gene-set enrichment analysis identified differential expression of genes in ER signaling pathways and DNA repair pathways in E2-treated cells. E2-induced ER signaling also increased {gamma}H2AX foci in 3 of the 4 cell lines. Levels of DSBs were increased by inhibition of the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. DSBs were also increased in MCF10A-ESR1 cells heterozygous for the BRCA1185delAG mutation causing a truncated protein. ConclusionsInducible expression of ER in immortalized HBECs recapitulate transcriptional, replicative and DNA damage responses. Increased DSBs in MCF10A-ESR1 cells with heterozygous mutation of BRCA1 indicate haploinsufficiency and the potential for increased genetic instability due to ER signaling.