Mechanism of and requirement for estrogen-regulated MYB expression in estrogen-receptor-positive breast cancer cells

Abstract

MYB (the human ortholog of c-myb) is expressed in a high proportion of human breast tumors, and that expression correlates strongly with estrogen receptor (ER) positivity. This may reflect the fact that MYB is a target of estrogen/ER signaling. Because in many cases MYB expression appears to be regulated by transcriptional attenuation or pausing in the first intron, we first investigated whether this mechanism was involved in estrogen/ER modulation of MYB. We found that this was the case and that estrogen acted directly to relieve attenuation due to sequences within the first intron, specifically, a region potentially capable of forming a stem-loop structure in the transcript and an adjacent poly(dT) tract. Secondly, given the involvement of MYB in hematopoietic and colon tumors, we also asked whether MYB was required for the proliferation of breast cancer cells. We found that proliferation of ER(+) but not ER(-) breast cancer cell lines was inhibited when MYB expression was suppressed by using either antisense oligonucleotides or RNA interference. Our results show that MYB is an effector of estrogen/ER signaling and provide demonstration of a functional role of MYB in breast cancer.

Fig. 1.

Estrogen induces increased MYB mRNA levels and overcomes transcriptional attenuation. (A) MCF-7 cells were treated with 10 nM β-estradiol for the indicated times in the presence or absence of 10 μg/ml cycloheximide, and the levels of MYB and pS2 mRNA were quantitated by quantitative RT-PCR. (B) Schematic representation of part of the human MYB gene illustrating the location of the SL and poly(dT) motifs (SL-dT) in the first intron and the location of probes (p1–p5) used in nuclear run-on transcription assays. (C) Transcriptional activity, measured by nuclear run-on assays, in the 5′ region of the MYB gene was compared before (T = 0) and after (T = 24) exposure of MCF-7 and MDA-MB-231 cells to 10 nM β-estradiol for 12 h. The α³²P-UTP-labeled RNA transcripts were hybridized to immobilized cRNA probes as indicated and detected by using a PhosphorImager.

Fig. 2.

Reporter assays reveal that β-estradiol relieves the suppression of transcription due to the SL–poly(dT) motif. (A) Schematic illustration of the MYB reporter construct in which CAT transcription is driven by the human MYB promoter through exon 1 and part of intron 1 (WT). Also shown are deletion constructs in which the entire SL–poly(dT) motif is deleted along with the remaining 3′ sequences of intron 1 (Δ3′ intron 1), only the SL motif is deleted (ΔSL), or only the poly(dT) motif is deleted (ΔΤ). (B) CAT activity (± SEM) in MCF-7 cells transiently transfected with the reporter constructs shown in A and cultured in the presence or absence, as indicated, of 10 nM β-estradiol for 12 h.

Fig. 3.

AS MYB oligonucleotide inhibits the proliferation of ER⁺ but not ER⁻ breast cancer cell lines. (A) Growth of MCF-7 cells cultured in the absence (no addition) or presence of 10 nM β-estradiol or cultured in the presence of β-estradiol and also treated with either 1 μM SCR or 1 μM MYB AS oligonucleotides. Mean cell numbers (± SD) of triplicate samples were determined daily as shown. (B) AS MYB oligonucleotide, but not the SCR oligonucleotide, decreases Myb protein levels in MCF-7 cells. Total cell lysates were collected for Western blot analysis with Myb and β-tubulin (as a loading control) antibodies. (C) The MYB AS oligonucleotide inhibits proliferation of ER⁺ but not ER⁻ breast cancer cell lines. The indicated cell lines were treated as in A, and the cell number was determined after 3 days. The results (± SEM) normalized to the cell numbers of β-estradiol-only-treated cultures of each cell line (set at 100%) are shown.

Fig. 4.

Knockdown of MYB by inducible shRNA decreases MCF-7 proliferation and Myb protein expression. (A) Schematic representation of the lentiviral vector pLVTSH showing the following key elements (left to right): LTR, H1 promoter, Tet operator, shRNA template, EF1α promoter, Tet repressor, internal ribosome entry site, GFP, self-inactivating LTR. (B and C) Parental MCF-7 cells or cells stably transduced with pLVTSH were grown in 10 nM β-estradiol in the absence or presence of Dox over 7 days. Neither the empty vector (Vector) nor a vector expressing a SCR affected proliferation, but the MYB shRNA vector inhibited proliferation when cells were grown in the presence of Dox. (D) Western blot analysis showing the decreased expression of Myb in only the MYB shRNA vector-transduced cells after treatment with Dox for 24 h. The membrane was also probed for β-tubulin as a loading control.

Fig. 5.

Knockdown of MYB with shRNA blocks cell cycle progression from the G₁/S phase in MCF-7 cells. The cell lines used in the studies illustrated in Fig. 4 were grown in the presence of β-estradiol with or without Dox, as indicated, for 72 h. They were then harvested for cell cycle distribution analysis by propidium iodide staining and flow cytometry; percentages of cells in G₀/G₁, S, and G₂/M are shown for each treatment group.