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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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Related in: MedlinePlus

NR2E3 maintains ESR1 function via direct binding to ESR1 promoter in breast cancer cellsA. Diagram of ESR1 gene promoter spanning from −245 to +212 bp, from −735 to +212 bp and from −2769 to +212 bp.B. MCF-7 cells transiently transfected with indicated ESR1 deletion reporters and NR2E3 construct.C. MCF-7 cells-stably knocked down by NR2E3 were transiently transfected with indicated ESR1 deletion constructs. Cell lysates were used for measuring the luciferase activity.D. Schematic representation of ESR1 promoter region for ChIP assay. Estrogen receptor response element (ERE).E. ChIP assay was done in MCF-7 or in T47D with NR2E3 antibody. Recruitment of NR2E3 to the ESR1 promoter was analysed using primers specific to the ESR1 promoter. IgG was used as an internal control.F-G. After stably transfecting control and NR2E3-specific shRNA in MCF-7 cells, the total viable cell numbers were determined by Coulter Z1 counter (Beckman Coulter, Brea, CA). Following stable transfection of shRNAs, the total viable cell numbers were determined after vehicle or E2 treatment with the indicated dose.H-I. Expression of CCND1 and TFF1 in shRNA transfected MCF-7 cells after vehicle or E2 treatment for 24 h. Student's t test (two-tailed) was applied to estimate the significance of gene expression changes. All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005). Significant differences in cells were compared with controls.
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fig04: NR2E3 maintains ESR1 function via direct binding to ESR1 promoter in breast cancer cellsA. Diagram of ESR1 gene promoter spanning from −245 to +212 bp, from −735 to +212 bp and from −2769 to +212 bp.B. MCF-7 cells transiently transfected with indicated ESR1 deletion reporters and NR2E3 construct.C. MCF-7 cells-stably knocked down by NR2E3 were transiently transfected with indicated ESR1 deletion constructs. Cell lysates were used for measuring the luciferase activity.D. Schematic representation of ESR1 promoter region for ChIP assay. Estrogen receptor response element (ERE).E. ChIP assay was done in MCF-7 or in T47D with NR2E3 antibody. Recruitment of NR2E3 to the ESR1 promoter was analysed using primers specific to the ESR1 promoter. IgG was used as an internal control.F-G. After stably transfecting control and NR2E3-specific shRNA in MCF-7 cells, the total viable cell numbers were determined by Coulter Z1 counter (Beckman Coulter, Brea, CA). Following stable transfection of shRNAs, the total viable cell numbers were determined after vehicle or E2 treatment with the indicated dose.H-I. Expression of CCND1 and TFF1 in shRNA transfected MCF-7 cells after vehicle or E2 treatment for 24 h. Student's t test (two-tailed) was applied to estimate the significance of gene expression changes. All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005). Significant differences in cells were compared with controls.

Mentions: To test whether regulation of ESR1 expression by NR2E3 is due to direct binding of NR2E3 on the ESR1 promoter region, we carried out the luciferase reporter assay to map the binding region of the ESR1 promoter using three promoter constructs containing different lengths of the ESR1 promoter as described (Fig 4A; deGraffenried et al, 2002). Transcription activities remained the same in all three constructs of ESR1 promoter (Fig 4B). In addition, transcription activity in shortest construct (−245 ESR1 Luc.) was diminished when expression of NR2E3 was silenced by shNR2E3 (Fig 4C). These results suggest that the binding sites reside near and/or inside of the −245 promoter region. The outcome of the reporter gene assay was supported by a subsequent ChIP assay. In agreement with reporter assay, NR2E3 only interact with ESR1 promoter between the −250 and +447 bp regions (Fig 4D and E). Taken together, our results strongly indicate that NR2E3 regulates ESR1 at the transcriptional level via direct binding to the ESR1 promoter.


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 maintains ESR1 function via direct binding to ESR1 promoter in breast cancer cellsA. Diagram of ESR1 gene promoter spanning from −245 to +212 bp, from −735 to +212 bp and from −2769 to +212 bp.B. MCF-7 cells transiently transfected with indicated ESR1 deletion reporters and NR2E3 construct.C. MCF-7 cells-stably knocked down by NR2E3 were transiently transfected with indicated ESR1 deletion constructs. Cell lysates were used for measuring the luciferase activity.D. Schematic representation of ESR1 promoter region for ChIP assay. Estrogen receptor response element (ERE).E. ChIP assay was done in MCF-7 or in T47D with NR2E3 antibody. Recruitment of NR2E3 to the ESR1 promoter was analysed using primers specific to the ESR1 promoter. IgG was used as an internal control.F-G. After stably transfecting control and NR2E3-specific shRNA in MCF-7 cells, the total viable cell numbers were determined by Coulter Z1 counter (Beckman Coulter, Brea, CA). Following stable transfection of shRNAs, the total viable cell numbers were determined after vehicle or E2 treatment with the indicated dose.H-I. Expression of CCND1 and TFF1 in shRNA transfected MCF-7 cells after vehicle or E2 treatment for 24 h. Student's t test (two-tailed) was applied to estimate the significance of gene expression changes. All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005). Significant differences in cells were compared with controls.
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Related In: Results  -  Collection

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fig04: NR2E3 maintains ESR1 function via direct binding to ESR1 promoter in breast cancer cellsA. Diagram of ESR1 gene promoter spanning from −245 to +212 bp, from −735 to +212 bp and from −2769 to +212 bp.B. MCF-7 cells transiently transfected with indicated ESR1 deletion reporters and NR2E3 construct.C. MCF-7 cells-stably knocked down by NR2E3 were transiently transfected with indicated ESR1 deletion constructs. Cell lysates were used for measuring the luciferase activity.D. Schematic representation of ESR1 promoter region for ChIP assay. Estrogen receptor response element (ERE).E. ChIP assay was done in MCF-7 or in T47D with NR2E3 antibody. Recruitment of NR2E3 to the ESR1 promoter was analysed using primers specific to the ESR1 promoter. IgG was used as an internal control.F-G. After stably transfecting control and NR2E3-specific shRNA in MCF-7 cells, the total viable cell numbers were determined by Coulter Z1 counter (Beckman Coulter, Brea, CA). Following stable transfection of shRNAs, the total viable cell numbers were determined after vehicle or E2 treatment with the indicated dose.H-I. Expression of CCND1 and TFF1 in shRNA transfected MCF-7 cells after vehicle or E2 treatment for 24 h. Student's t test (two-tailed) was applied to estimate the significance of gene expression changes. All results are shown as mean plus standard deviation (SD) from three-independent replicates (*p < 0.05, **p < 0.01 and ***p < 0.005). Significant differences in cells were compared with controls.
Mentions: To test whether regulation of ESR1 expression by NR2E3 is due to direct binding of NR2E3 on the ESR1 promoter region, we carried out the luciferase reporter assay to map the binding region of the ESR1 promoter using three promoter constructs containing different lengths of the ESR1 promoter as described (Fig 4A; deGraffenried et al, 2002). Transcription activities remained the same in all three constructs of ESR1 promoter (Fig 4B). In addition, transcription activity in shortest construct (−245 ESR1 Luc.) was diminished when expression of NR2E3 was silenced by shNR2E3 (Fig 4C). These results suggest that the binding sites reside near and/or inside of the −245 promoter region. The outcome of the reporter gene assay was supported by a subsequent ChIP assay. In agreement with reporter assay, NR2E3 only interact with ESR1 promoter between the −250 and +447 bp regions (Fig 4D and E). Taken together, our results strongly indicate that NR2E3 regulates ESR1 at the transcriptional level via direct binding to the ESR1 promoter.

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