Estrogen Receptor α Controls a Gene Network in Luminal-Like Breast Cancer Cells Comprising Multiple Transcription Factors and MicroRNAs

Abstract

Luminal-like breast tumor cells express estrogen receptor ␣ (ER␣),

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer

“…It is worth mentioning that the cell cycle inhibitory effects of ERb are well known (Heldring et al, 2007;Grober et al, 2011, and references therein) and are more evident at relatively higher concentrations of E2 (X10 À10 ), compatible with the lower affinity of this ER subtype for the hormone (compare, for each cell clone, the S þ G2 fraction in hormone-stimulated vs -starved cells). The efficiency of PPT in promoting cell cycle progression (Figure 1d) relates to its ability to promote ERamediated gene transcription (Figure 1b), confirming the direct link between transcriptional activity of this receptor subtype and the mitogenic effects of estrogen (Cicatiello et al, 2010). Gene-expression profiling of asynchronously growing cells showed no major differences between N-and C-TAP-ERb cells, whereas their transcriptomes were significantly different from that of C-TAP-ERa cells (Supplementary Figure S1), confirming previous results obtained in E2-stimulated cells (Grober et al, 2011).…”

“…The effects of ERb in regulation of the first step in miRNA biogenesis were investigated by measuring changes in pri-miR expression in control (wt), N-TAP-and C-TAP-ERb cells before and after E2 stimulation. Results show that in the absence of ERb, estrogen stimulation did not influence primary-transcript levels, assessed by both quantitative real-time rtPCR and RNA-expression profiling (c9orf3 RNA; Figure 4b and Cicatiello et al, 2010). On the other hand, a slight but reproducible accumulation of pri-miR-23b/ -27b/-24-1 was detectable in ERb þ cells already 2 h after E2 (Figure 4b).…”

“…We analyzed by chromatin immunoprecipitation sequencing (ChIPSeq) the entire ERa and ERb cistromes in the ERb þ (Grober et al, 2011) and ERbÀ cells (Cicatiello et al, 2010) upon E2 stimulation. Aligning ER-binding sites and miRNA gene positioning in the genome we observed that several miRNA-encoding genes differentially expressed in ERb þ vs ERbÀ cell lines (Supplementary Table S3A) and/or mammary tumors Table S3B) display ER-binding sites within 10 kb of the transcription unit, including sites where both ERs can be found together, likely associated in heterodimers.…”

“…Cells were hormone deprived for 4 days and chromatin was extracted from replicate samples before (ÀE2) or 45 min after stimulation with E2 as described previously (Cicatiello et al, 2010;Grober et al, 2011). Chromatin samples were incubated at 4 1C overnight with Abs against the C-(HC-20, from Santa Cruz Biotechnology, Europe) or N-(18-32, Sigma Aldrich Italia, Milano, Italy) terminus of human ERa, anti-Drosha (ab12286, Abcam, used as described by Nakamura et al (2007), anti-DDX5 (ab21696, Abcam) or, for TAP-ERb, with IgG Sepharose 6 fast Flow (GE Healthcare) as described earlier (Grober et al, 2011).…”

“…Several evidences indicate that ERa is among the transcription factors regulating miRNA biogenesis in hormone-responsive BC cells (Bhat-Nakshatri et al, 2009;Castellano et al, 2009;Maillot et al, 2009;Yamagata et al, 2009;Cicatiello et al, 2010;Ferraro et al, 2010Ferraro et al, , 2011. More recently, global mapping of ERb binding to ERa-positive, hormone-responsive BC cells chromatin in vivo showed ERb interaction with several miRNA genes, suggesting the possible involvement of this receptor in hormonal control of small noncoding RNA biogenesis in this cell type (Grober et al, 2011).…”