Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage
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Background
Signaling by estrogen-receptor alpha (ERα) plays a major role in breast cancer initiation Investigations of the mechanism of DNA damage mediated by ERα signaling are carried out in breast cancer cell lines due to the lack of ERα+ normal human breast epithelial cells lines (HBEC). Defining the mechanisms by which ERα induces DNA damage and initiates tumorigenesis requires normal HBECs that express ERα, demonstrate estrogenic responses, and are amenable to long term propagation in culture.
Methods
We utilized lentiviral expression of an inducible ERα construct to generate four HBEC lines (HBEC- ESR1 ). We studied these cells for ERα-dependent responses using a luciferase reporter, endogenous gene expression and proliferation assays. RNA-Seq was performed to characterize the ERα-mediated transcriptomic patterns in the four HBEC lines. ERα mediated DNA double strand breaks (DSBs) were analyzed using γH2AX immunofluorescence.
Results
Expression and functional activation of ERα were observed in all HBEC- ESR1 lines, whereas proliferation in response to 17β-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 ERα+ breast cancer cell lines (MCF7 or T47D). Gene-set enrichment analysis identified differential expression of genes in ERα signaling pathways and DNA repair pathways in E2-treated cells. E2-induced ERα signaling also increased γH2AX 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 BRCA1 185delAG mutation causing a truncated protein.
Conclusions
Inducible expression of ERα 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 ERα signaling.
