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Reconstruction of nuclear receptor network reveals that NR2E3 is a novel upstream regulator of ESR1 in breast cancer.

Park YY, Kim K, Kim SB, Hennessy BT, Kim SM, Park ES, Lim JY, Li J, Lu Y, Gonzalez-Angulo AM, Jeong W, Mills GB, Safe S, Lee JS - EMBO Mol Med (2011)

Bottom Line: By applying systems-level re-analysis of publicly available gene expression data, we uncovered a potential regulator of ESR1.Moreover, expression of NR2E3 was significantly associated with recurrence-free survival and a favourable response to tamoxifen treatment in women with ER-positive breast cancer.Our results provide mechanistic insights on the regulation of ESR1 by NR2E3 and the clinical relevance of NR2E3 in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

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NR2E3 regulates ESR1 function in breast cancer cellsA-B. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe.C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes.E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody.F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity.G-H. MCF-7 cells were transiently transfected with indicated siRNA, and cell lysates were used for Western blot (G) or for qRT-PCR (H). All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005).
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fig03: NR2E3 regulates ESR1 function in breast cancer cellsA-B. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe.C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes.E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody.F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity.G-H. MCF-7 cells were transiently transfected with indicated siRNA, and cell lysates were used for Western blot (G) or for qRT-PCR (H). All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005).

Mentions: Since little is known about the function of NR2E3 in breast cancer, we investigated possible roles of NR2E3 related to the ESR1-signalling pathway using NR2E3-specific small hairpin RNA (shRNA) in estrogen receptor (ER)-positive breast cancer MCF-7 cells (Fig 3A). Surprisingly, when expression of NR2E3 was silenced by shRNA, expression of ESR1 and its downstream targets (GATA3, PGR, CCND1 and TFF1) were also significantly downregulated (Fig 3B); reduced expression of ESR1 and its downstream targets was also validated at the protein level (Fig 3E). The effect of silencing NR2E3 expression on ESR1 and its downstream targets was also highly reproducible in another ER-positive breast cancer cell line: T47D (Fig 3C and D). It is interesting to point out that expression of FOXA1 was not altered after silencing NR2E3expression in MCF-7 cells while its expression was down-regulated in T47D cells, suggesting that additional regulatory mechanisms for expression of FOXA1 might exist in MCF-7 cells. Transcriptional activity of ESR1 was also diminished by small hairpin NR2E3 (shNR2E3; Fig 3F). Furthermore, overexpression of exogenous NR2E3 further increased expression of ESR1 and its downstream targets as well as its transcriptional activity in MCF-7 cells (Fig S1 of Supporting Information), strongly demonstrating that NR2E3 regulates ESR1 expression and subsequent ESR1-mediated induction of target genes.


Reconstruction of nuclear receptor network reveals that NR2E3 is a novel upstream regulator of ESR1 in breast cancer.

Park YY, Kim K, Kim SB, Hennessy BT, Kim SM, Park ES, Lim JY, Li J, Lu Y, Gonzalez-Angulo AM, Jeong W, Mills GB, Safe S, Lee JS - EMBO Mol Med (2011)

NR2E3 regulates ESR1 function in breast cancer cellsA-B. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe.C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes.E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody.F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity.G-H. MCF-7 cells were transiently transfected with indicated siRNA, and cell lysates were used for Western blot (G) or for qRT-PCR (H). All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005).
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Related In: Results  -  Collection

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fig03: NR2E3 regulates ESR1 function in breast cancer cellsA-B. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total RNA from indicated cell extracted and analysed by qRT-PCR with indicated probe.C-D. T47D cells were stably transfected with shNR2E3 or shCon. Total RNA and protein from indicated cell extracts were analysed by qRT-PCR with indicated probes.E. MCF-7 cells were stably transfected with shNR2E3 or control shRNA (shCon). Total protein from indicated cell extracted and analysed by Western blot with indicated antibody.F. Stably transfected MCF-7 with shNR2E3 or with shCon were transfected with ESR1 promoter construct, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity.G-H. MCF-7 cells were transiently transfected with indicated siRNA, and cell lysates were used for Western blot (G) or for qRT-PCR (H). All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005).
Mentions: Since little is known about the function of NR2E3 in breast cancer, we investigated possible roles of NR2E3 related to the ESR1-signalling pathway using NR2E3-specific small hairpin RNA (shRNA) in estrogen receptor (ER)-positive breast cancer MCF-7 cells (Fig 3A). Surprisingly, when expression of NR2E3 was silenced by shRNA, expression of ESR1 and its downstream targets (GATA3, PGR, CCND1 and TFF1) were also significantly downregulated (Fig 3B); reduced expression of ESR1 and its downstream targets was also validated at the protein level (Fig 3E). The effect of silencing NR2E3 expression on ESR1 and its downstream targets was also highly reproducible in another ER-positive breast cancer cell line: T47D (Fig 3C and D). It is interesting to point out that expression of FOXA1 was not altered after silencing NR2E3expression in MCF-7 cells while its expression was down-regulated in T47D cells, suggesting that additional regulatory mechanisms for expression of FOXA1 might exist in MCF-7 cells. Transcriptional activity of ESR1 was also diminished by small hairpin NR2E3 (shNR2E3; Fig 3F). Furthermore, overexpression of exogenous NR2E3 further increased expression of ESR1 and its downstream targets as well as its transcriptional activity in MCF-7 cells (Fig S1 of Supporting Information), strongly demonstrating that NR2E3 regulates ESR1 expression and subsequent ESR1-mediated induction of target genes.

Bottom Line: By applying systems-level re-analysis of publicly available gene expression data, we uncovered a potential regulator of ESR1.Moreover, expression of NR2E3 was significantly associated with recurrence-free survival and a favourable response to tamoxifen treatment in women with ER-positive breast cancer.Our results provide mechanistic insights on the regulation of ESR1 by NR2E3 and the clinical relevance of NR2E3 in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Show MeSH
Related in: MedlinePlus