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. 2012 Nov 30;287(49):41514-22.
doi: 10.1074/jbc.M112.404871. Epub 2012 Oct 11.

Estrogen receptor α signaling regulates breast tumor-initiating cells by down-regulating miR-140 which targets the transcription factor SOX2

Yongshu Zhang  1 , Gabriel Eades, Yuan Yao, Qinglin Li, Qun Zhou
Affiliations

Estrogen receptor α signaling regulates breast tumor-initiating cells by down-regulating miR-140 which targets the transcription factor SOX2

Yongshu Zhang et al. J Biol Chem. .

Abstract

Several reports have indicated that miR-140, a possible tumor suppressor microRNA (miR), is down-regulated in breast tumors compared with normal breast tissues. However, the role of miR-140 in breast tumorigenesis is unclear. We initiated studies that examined estrogen receptor α (ERα) signaling in the tissue-specific regulation of miR-140 in breast cancer. We found that estrogen stimulation of ERα-positive breast cancer cells resulted in decreased miR-140 expression. We performed promoter analyses and examined predicted ERα binding elements in the miR-140 promoter using luciferase constructs of a miR-140 promoter deletion series. Our studies revealed that ERα binds to one specific estrogen response element flanking the miR-140 promoter and consequently suppresses miR-140 transcription. We found that the stem cell self-renewal regulator SOX2 is a novel target of miR-140, and that this miR-140/SOX2 pathway critically regulates breast tumor-initiating cell survival, providing a new link between ERα signaling and breast cancer stem cell maintenance.

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Figures

FIGURE 1.
FIGURE 1.
The transcription factor SOX2 is a novel target for miR-140 in breast cancer cells. A, targetScan 6.0 predicts a miR-140 response element in the SOX2 mRNA 3′-UTR. An illustration of the SOX2 3′-UTR as well as the seed sequence of miR-140. B, inverse expression of miR-140 and SOX2. Relative expression of miR-140 and SOX2 in MCF10A and MCF-7 cells as determined by qRT-PCR. C, (left) HEK-293T cells were transfected with 2 μg pGL3 luciferase vector containing either wild-type SOX2 3′-UTR or mutant SOX2 3′-UTR (abolishing miR-140 targeting). Cells were co-transfected along with 2 μg miR-140 expression vector for 48h and lysed and luciferase activity was measured with a luminometer. Right, MCF-7 cells were transfected with miR-140 expression vector and SOX2 mRNA was measured by qRT-PCR, normalizing to GAPDH mRNA. D, MCF10A cells were transfected with 2 μg of miR-140 sponge inhibitor for 24 h, and SOX2 protein was measured by Western blot, normalizing to β-actin expression. n = 3, mean ± S.E.
FIGURE 2.
FIGURE 2.
Estrogen receptor signaling regulates miR-140 and SOX2 expression in normal breast tissue and in breast cancer cells. A, Western blot showing ERα expression in non-tumorigenic mammary epithelial cells (MCF10A) and MCF10A cells stably transfected with ERα (MCF10AERIN) cells, normalizing to β-actin expression. ERα-positive MCF-7 breast cancer cells are included as a control. B, MCF10A and MCF10AERIN were treated with 10 nm estradiol (E2) or EtOH control for 24 h. miR-140 and SOX2 expression was measured by qRT-PCR normalizing to U6 snRNA or GAPDH mRNA, respectively. C, MCF-7 cells were treated with 10 nm E2 for 24 h and miR-140 and SOX2 expression was measured by qRT-PCR. Pretreatment with 50 nm ERα siRNA was used to block E2-induced changes in mir-140 expression. n = 3 ± S.E. D, immunohistochemistry staining of normal breast tissue and breast tumor tissue (IDC associated with DCIS) with SOX2 and ERα antibody. E, qRT-PCR analysis of miR-140 expression in normal breast tissue and fresh tumor tissue from patients with IDC, normalizing to U6 snRNA.
FIGURE 3.
FIGURE 3.
A, identification of Estrogen Response Elements (ERE) in the miR-140 Promoter. The miR-140 promoter region, ∼2 kb of the region upstream of the TSS of Wwp2 gene, was cloned into a luciferase reporter to examine promoter activity. Predicted ERE sites (3) in the miR-140 region were mutated or deleted in a series of truncated reporters. MCF-7 cells were transfected with 2 μg of reporter plasmid along with control Renilla luciferase reporter for dual-luciferase activity assays. Cells were treated with 10 nm E2 or EtOH vehicle control. After 24 h cells were lysed and assayed for luciferase activity. Control reporter possessed no promoter activity. n = 3 ± S.E. B, estrogen stimulation enhances ERα binding at the miR-140 promoter. Results for miR-140 promoter region ChIP for ERα antibody. Cross-linked, sonicated chromatin was immunoprecipitated with ERα antibody or negative control rabbit IgG antibody following stimulation with 10 nm E2 or 10 nm BPA or EtOH control. qRT-PCR was carried out for miR-140 promoter region (−79/−50 ERE). n = 2, mean ± S.E.
FIGURE 4.
FIGURE 4.
A, SOX2 is required for tumor-initiating cell survival in MCF-7 breast cancer cells. MCF-7 breast cancer cells were stably transfected with SOX2 shRNA (followed by puromycin selection) or transiently transfected with 2 μg of SOX2 expression construct for 24 h. Cells were stained with CD44-APC and CD24-PE antibodies and CD44+/CD24- subpopulations were examined by flow cytometry. n = 3. B, qRT-PCR showing SOX2 mRNA and ERα mRNA levels following SOX2 overexpression or knockdown or miR-140 overexpression, normalizing to GAPDH mRNA.
FIGURE 5.
FIGURE 5.
The miR-140/SOX2 pathway regulates mammosphere formation of breast cancer cells. A, MCF-7 cells were transfected with 2 μg of miR-140 expression vector, 2 μg of SOX2 expression construct, or co-transfected with both. Transfected MCF-7 cells were collected using non-enzymatic dissociation buffer, separated to single cells by passing through 40 μm cell strainer and seeded at 20,000 cells/ml on attachment free 6-well plates coated with 2% poly-HEMA. After 7 days, mammospheres greater than 100 μm were counted. For subsequent passages, mammospheres were collected, separated into single cells, and re-seeded at 10,000 cells/ml. Pictures taken of primary passage mammospheres at 7 days are shown. B, average results from first and second passage mammosphere experiments were quantified and represented in bar graphs. n = 3, mean ± S.E. p value determined by Student's t test, *, p < 0.05. #, not significant.
FIGURE 6.
FIGURE 6.
miR-140 regulates tumor-initiating cell renewal in estrogen-stimulated breast cancer cells. A, MCF-7 cells were cultured in starvation conditions in medium lacking phenol red and charcoal stripped serum. Cells were treated with 10 nm E2 or vehicle control and transfected with 2 μg of miR-140 expression vector or control vector. After 24 h, cells were stained with CD44-APC and CD24-PE antibodies, and CD44+/CD24- subpopulations were examined by flow cytometry. B, T47D cells were cultured in starvation conditions, treated with 10 nm E2, and transfected with 2 μg of miR-140 expression vector. Following 24 h CD44+/CD24- subpopulation frequency was examined by flow cytometry. n = 2.
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