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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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PIAS3 association with ESR1 via NR2E3A-B. MCF-7 breast cancer cells were transiently transfected with NCOA1, 2 and 3 specific siRNA Smart Pool or control siLuc. Protein lysates were isolated from indicated samples and used for Western blot with indicated antibodies (A) and 30 ng of total RNA from transfected cell lines were analysed by qRT-PCR using gene-specific primers as indicated (B). Student t-test (two-tailed) was applied to estimate the significance of gene expression changes: ***p < 0.005, **p < 0.01 and *p < 0.05.C-D. MCF-7 cells were transiently transfected with siPIAS3 or siLuc. Total RNA or protein from indicated cell extracts was analysed by qRT-PCR (C) or Western blot (D) to detect the indicated mRNA or protein expression levels.E. MCF-7 cells were transiently transfected with siESR1 or siLuc. Protein from indicated cell extracts was analysed by Western blot to detect the indicated protein expression levels.F.ESR1 promoter construct was transfected with indicated constructs after indicated siRNA was transfected with MCF-7 cells, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. Western blot shows silencing efficiency of UBC9.G. Co-IP of NR2E3 with PIAS3 analysed by Western blotting from MCF-7 cells.H. GST-NR2E3 was incubated with His-PIAS3 for 2 h at 4°C and then isolated from reaction mixture by an immobilized nickel resin. The resulting precipitates were subjected to immunoblot analysis to NR2E3 or PIAS3.I. ChIP assay was done in MCF-7 with PIAS3 or NR2E3 antibodies. Recruitment of PIAS3 to the ESR1 promoter was analysed using a primer specific to the ESR1 promoter. IgG was used as an internal control.J. After MCF-7 cells were transiently transfected with siPIAS3 or siLuc., cells were used for ChIP assay.
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fig05: PIAS3 association with ESR1 via NR2E3A-B. MCF-7 breast cancer cells were transiently transfected with NCOA1, 2 and 3 specific siRNA Smart Pool or control siLuc. Protein lysates were isolated from indicated samples and used for Western blot with indicated antibodies (A) and 30 ng of total RNA from transfected cell lines were analysed by qRT-PCR using gene-specific primers as indicated (B). Student t-test (two-tailed) was applied to estimate the significance of gene expression changes: ***p < 0.005, **p < 0.01 and *p < 0.05.C-D. MCF-7 cells were transiently transfected with siPIAS3 or siLuc. Total RNA or protein from indicated cell extracts was analysed by qRT-PCR (C) or Western blot (D) to detect the indicated mRNA or protein expression levels.E. MCF-7 cells were transiently transfected with siESR1 or siLuc. Protein from indicated cell extracts was analysed by Western blot to detect the indicated protein expression levels.F.ESR1 promoter construct was transfected with indicated constructs after indicated siRNA was transfected with MCF-7 cells, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. Western blot shows silencing efficiency of UBC9.G. Co-IP of NR2E3 with PIAS3 analysed by Western blotting from MCF-7 cells.H. GST-NR2E3 was incubated with His-PIAS3 for 2 h at 4°C and then isolated from reaction mixture by an immobilized nickel resin. The resulting precipitates were subjected to immunoblot analysis to NR2E3 or PIAS3.I. ChIP assay was done in MCF-7 with PIAS3 or NR2E3 antibodies. Recruitment of PIAS3 to the ESR1 promoter was analysed using a primer specific to the ESR1 promoter. IgG was used as an internal control.J. After MCF-7 cells were transiently transfected with siPIAS3 or siLuc., cells were used for ChIP assay.

Mentions: Many biochemical and genetic studies have demonstrated that coregulators are critically important for the function of NRs and the induction of NR-dependent genes (Xu et al, 2009). Since NR coactivators (NCOAs, also known as steroid receptor coactivators) are the best-known coregulators of NRs and interact with diverse NRs in human cancer (Xu et al, 2009), we first investigated their possible role in regulating ESR1 expression by NR2E3. After silencing NCOA1, 2 and 3 by their specific siRNA (SMART Pool) in MCF-7 cells, we measured gene expression of ESR1 and its downstream target genes. While silencing the expression of the NCOA family genes significantly downregulated expression of ESR1 downstream target genes, expression of ESR1 itself was not altered (Fig 5A and B). When we used different siRNAs to knock down NCOA family members, the result were significant as shown in Fig S2B and C of Supporting Information. This suggests that the NCOAs are only involved in regulation of ESR1's transcriptional activity and do not influence NR2E3-mediated regulation of ESR1 expression in breast cancer cells.


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)

PIAS3 association with ESR1 via NR2E3A-B. MCF-7 breast cancer cells were transiently transfected with NCOA1, 2 and 3 specific siRNA Smart Pool or control siLuc. Protein lysates were isolated from indicated samples and used for Western blot with indicated antibodies (A) and 30 ng of total RNA from transfected cell lines were analysed by qRT-PCR using gene-specific primers as indicated (B). Student t-test (two-tailed) was applied to estimate the significance of gene expression changes: ***p < 0.005, **p < 0.01 and *p < 0.05.C-D. MCF-7 cells were transiently transfected with siPIAS3 or siLuc. Total RNA or protein from indicated cell extracts was analysed by qRT-PCR (C) or Western blot (D) to detect the indicated mRNA or protein expression levels.E. MCF-7 cells were transiently transfected with siESR1 or siLuc. Protein from indicated cell extracts was analysed by Western blot to detect the indicated protein expression levels.F.ESR1 promoter construct was transfected with indicated constructs after indicated siRNA was transfected with MCF-7 cells, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. Western blot shows silencing efficiency of UBC9.G. Co-IP of NR2E3 with PIAS3 analysed by Western blotting from MCF-7 cells.H. GST-NR2E3 was incubated with His-PIAS3 for 2 h at 4°C and then isolated from reaction mixture by an immobilized nickel resin. The resulting precipitates were subjected to immunoblot analysis to NR2E3 or PIAS3.I. ChIP assay was done in MCF-7 with PIAS3 or NR2E3 antibodies. Recruitment of PIAS3 to the ESR1 promoter was analysed using a primer specific to the ESR1 promoter. IgG was used as an internal control.J. After MCF-7 cells were transiently transfected with siPIAS3 or siLuc., cells were used for ChIP assay.
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fig05: PIAS3 association with ESR1 via NR2E3A-B. MCF-7 breast cancer cells were transiently transfected with NCOA1, 2 and 3 specific siRNA Smart Pool or control siLuc. Protein lysates were isolated from indicated samples and used for Western blot with indicated antibodies (A) and 30 ng of total RNA from transfected cell lines were analysed by qRT-PCR using gene-specific primers as indicated (B). Student t-test (two-tailed) was applied to estimate the significance of gene expression changes: ***p < 0.005, **p < 0.01 and *p < 0.05.C-D. MCF-7 cells were transiently transfected with siPIAS3 or siLuc. Total RNA or protein from indicated cell extracts was analysed by qRT-PCR (C) or Western blot (D) to detect the indicated mRNA or protein expression levels.E. MCF-7 cells were transiently transfected with siESR1 or siLuc. Protein from indicated cell extracts was analysed by Western blot to detect the indicated protein expression levels.F.ESR1 promoter construct was transfected with indicated constructs after indicated siRNA was transfected with MCF-7 cells, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. Western blot shows silencing efficiency of UBC9.G. Co-IP of NR2E3 with PIAS3 analysed by Western blotting from MCF-7 cells.H. GST-NR2E3 was incubated with His-PIAS3 for 2 h at 4°C and then isolated from reaction mixture by an immobilized nickel resin. The resulting precipitates were subjected to immunoblot analysis to NR2E3 or PIAS3.I. ChIP assay was done in MCF-7 with PIAS3 or NR2E3 antibodies. Recruitment of PIAS3 to the ESR1 promoter was analysed using a primer specific to the ESR1 promoter. IgG was used as an internal control.J. After MCF-7 cells were transiently transfected with siPIAS3 or siLuc., cells were used for ChIP assay.
Mentions: Many biochemical and genetic studies have demonstrated that coregulators are critically important for the function of NRs and the induction of NR-dependent genes (Xu et al, 2009). Since NR coactivators (NCOAs, also known as steroid receptor coactivators) are the best-known coregulators of NRs and interact with diverse NRs in human cancer (Xu et al, 2009), we first investigated their possible role in regulating ESR1 expression by NR2E3. After silencing NCOA1, 2 and 3 by their specific siRNA (SMART Pool) in MCF-7 cells, we measured gene expression of ESR1 and its downstream target genes. While silencing the expression of the NCOA family genes significantly downregulated expression of ESR1 downstream target genes, expression of ESR1 itself was not altered (Fig 5A and B). When we used different siRNAs to knock down NCOA family members, the result were significant as shown in Fig S2B and C of Supporting Information. This suggests that the NCOAs are only involved in regulation of ESR1's transcriptional activity and do not influence NR2E3-mediated regulation of ESR1 expression in breast cancer cells.

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