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The BCL9-2 proto-oncogene governs estrogen receptor alpha expression in breast tumorigenesis.

Zatula N, Wiese M, Bunzendahl J, Birchmeier W, Perske C, Bleckmann A, Brembeck FH - Oncotarget (2014)

Bottom Line: BCL9-2 overexpression in vivo delayed the mammary involution and induced alveolar hyperplasia.We found, that primary cell cultures of BCL9-2 breast tumors responded to tamoxifen treatment.Finally, we describe a novel mechanism, how BCL9-2 regulates ER transcription by interaction with Sp1 through the proximal ESR1 gene promoter.

View Article: PubMed Central - PubMed

Affiliation: Tumor Biology and Signal Transduction, Georg-August-University Göttingen, Germany. Dept. of Hematology and Medical Oncology, Georg-August-University Göttingen, Germany.

ABSTRACT
The majority of human breast cancers express estrogen receptor alpha (ER), which is important for therapy with anti-estrogens. Here we describe the role of BCL9-2, a proto-oncogene previously characterized as co-activator of Wnt/ß-catenin signaling, for mammary tumorigenesis in mice and human. ER positive human breast cancers showed overexpression of BCL9-2 and tamoxifen treated patients with high BCL9-2 demonstrated a better survival. BCL9-2 was upregulated during puberty and pregnancy in normal mammary epithelia, but downregulated in the involuted gland. BCL9-2 overexpression in vivo delayed the mammary involution and induced alveolar hyperplasia. Moreover, aged BCL9-2 transgenic mice developed ductal-like mammary tumors with high nuclear ER expression. We found, that primary cell cultures of BCL9-2 breast tumors responded to tamoxifen treatment. Moreover, BCL9-2 regulated the expression of ER and the proliferation of human breast cancer cells independently of ß-catenin. Finally, we describe a novel mechanism, how BCL9-2 regulates ER transcription by interaction with Sp1 through the proximal ESR1 gene promoter. In summary, BCL9-2 induces ER positive breast cancers in vivo, regulates ER expression by a novel ß-catenin independent mechanism in breast cancer cells, and might predict the therapy response to tamoxifen treatment.

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BCL9-2 regulates the transcription of ER in the proximal promoter and interacts with Sp1 in human breast cancer cells(A) Schematic view of regulatory elements within the upstream sequence of the human ESR1 gene that are important for ESR1 gene transcription in breast cancer cells (according to Kos et al., 2001). One previously described G/C-box in promoter B and a G/C-rich element in promoter A as potential Sp1 binding sites are also indicated. The amplified promoter regions by conventional PCR and qRT-PCR following ChIP are indicated below the scheme. (B) Representative examples of the PCR analysis for the indicated promoter fragments. ChIP experiments were performed for the indicated promoter regions of the ESR-1 gene and the GAPDH promoter as control. (C) Re-ChIP experiments of BCL9-2 and Sp1 for the two most proximal promoter regions of the ESR1 gene in MCF7. Representative examples of the PCR analysis following ChIP and re-ChIP with the indicated antibodies and IgG controls are shown. Rabbit IgG (rb IgG) was used as negative control. (D) qRT-PCR analyses for the indicated promoter regions of at least three independent ChIP experiments in MCF7 following immunoprecipitation of the DNA with the indicated antibodies. We performed the absolute quantification using the regression analysis method and calculated the values as percent of input DNA after normalization to the control IgG. (E) qRT-PCR of at least three ChIP experiments following mithramycin A treatment. MCF7 cells were treated with 10 nM mithramycin A or vehicle (0.1% EtOH) for 24 hours prior to fixation. The fold enrichment was calculated using the regression analysis method and normalized to control IgG. (F) Luciferase activity of a reporter containing the most proximal ESR1 gene promoter (WT = wild type), of a G/C box mutant in promoter B (MT-B) and of a G/C-rich element in promoter A (MT-A) as indicated in the scheme. The graph shows a representative dose response experiment performed in triplicates and their range. HEK293 cells were transfected with 100 ng reporter constructs and 0, 12.5, 50 und 100 ng BCL9-2 expression constructs for 72 hours. (G) Western Blots (WB) analyses of MCF7 cell lysates after co-immunoprecipitation (IP) with the indicated antibodies. As negative controls, mouse and rabbit IgG's were used.
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Figure 7: BCL9-2 regulates the transcription of ER in the proximal promoter and interacts with Sp1 in human breast cancer cells(A) Schematic view of regulatory elements within the upstream sequence of the human ESR1 gene that are important for ESR1 gene transcription in breast cancer cells (according to Kos et al., 2001). One previously described G/C-box in promoter B and a G/C-rich element in promoter A as potential Sp1 binding sites are also indicated. The amplified promoter regions by conventional PCR and qRT-PCR following ChIP are indicated below the scheme. (B) Representative examples of the PCR analysis for the indicated promoter fragments. ChIP experiments were performed for the indicated promoter regions of the ESR-1 gene and the GAPDH promoter as control. (C) Re-ChIP experiments of BCL9-2 and Sp1 for the two most proximal promoter regions of the ESR1 gene in MCF7. Representative examples of the PCR analysis following ChIP and re-ChIP with the indicated antibodies and IgG controls are shown. Rabbit IgG (rb IgG) was used as negative control. (D) qRT-PCR analyses for the indicated promoter regions of at least three independent ChIP experiments in MCF7 following immunoprecipitation of the DNA with the indicated antibodies. We performed the absolute quantification using the regression analysis method and calculated the values as percent of input DNA after normalization to the control IgG. (E) qRT-PCR of at least three ChIP experiments following mithramycin A treatment. MCF7 cells were treated with 10 nM mithramycin A or vehicle (0.1% EtOH) for 24 hours prior to fixation. The fold enrichment was calculated using the regression analysis method and normalized to control IgG. (F) Luciferase activity of a reporter containing the most proximal ESR1 gene promoter (WT = wild type), of a G/C box mutant in promoter B (MT-B) and of a G/C-rich element in promoter A (MT-A) as indicated in the scheme. The graph shows a representative dose response experiment performed in triplicates and their range. HEK293 cells were transfected with 100 ng reporter constructs and 0, 12.5, 50 und 100 ng BCL9-2 expression constructs for 72 hours. (G) Western Blots (WB) analyses of MCF7 cell lysates after co-immunoprecipitation (IP) with the indicated antibodies. As negative controls, mouse and rabbit IgG's were used.

Mentions: Since BCL9-2 lacks a conserved DNA binding domain, we included transcription factors that were previously described to bind within the ESR1 gene promoter and activate ER transcription in breast cancer cells (Fig. 7A). First, we analyzed three larger regions in the upstream DNA sequence of the ESR1 gene by conventional PCR following ChIP. The first fragment covered the most proximal promoter A [based on the nomenclature by 33] that is occupied by a complex containing Sp1, p53 and other factors of the basal transcription machinery [7]. Although the binding sites for Sp1 in promoter A have not been described yet, our analyses using Transfac Patch tool [34] or JASPAR database [35] revealed that the ESR1 gene promoter A contains multiple G/C-rich sequences surrounding the transcription start site.


The BCL9-2 proto-oncogene governs estrogen receptor alpha expression in breast tumorigenesis.

Zatula N, Wiese M, Bunzendahl J, Birchmeier W, Perske C, Bleckmann A, Brembeck FH - Oncotarget (2014)

BCL9-2 regulates the transcription of ER in the proximal promoter and interacts with Sp1 in human breast cancer cells(A) Schematic view of regulatory elements within the upstream sequence of the human ESR1 gene that are important for ESR1 gene transcription in breast cancer cells (according to Kos et al., 2001). One previously described G/C-box in promoter B and a G/C-rich element in promoter A as potential Sp1 binding sites are also indicated. The amplified promoter regions by conventional PCR and qRT-PCR following ChIP are indicated below the scheme. (B) Representative examples of the PCR analysis for the indicated promoter fragments. ChIP experiments were performed for the indicated promoter regions of the ESR-1 gene and the GAPDH promoter as control. (C) Re-ChIP experiments of BCL9-2 and Sp1 for the two most proximal promoter regions of the ESR1 gene in MCF7. Representative examples of the PCR analysis following ChIP and re-ChIP with the indicated antibodies and IgG controls are shown. Rabbit IgG (rb IgG) was used as negative control. (D) qRT-PCR analyses for the indicated promoter regions of at least three independent ChIP experiments in MCF7 following immunoprecipitation of the DNA with the indicated antibodies. We performed the absolute quantification using the regression analysis method and calculated the values as percent of input DNA after normalization to the control IgG. (E) qRT-PCR of at least three ChIP experiments following mithramycin A treatment. MCF7 cells were treated with 10 nM mithramycin A or vehicle (0.1% EtOH) for 24 hours prior to fixation. The fold enrichment was calculated using the regression analysis method and normalized to control IgG. (F) Luciferase activity of a reporter containing the most proximal ESR1 gene promoter (WT = wild type), of a G/C box mutant in promoter B (MT-B) and of a G/C-rich element in promoter A (MT-A) as indicated in the scheme. The graph shows a representative dose response experiment performed in triplicates and their range. HEK293 cells were transfected with 100 ng reporter constructs and 0, 12.5, 50 und 100 ng BCL9-2 expression constructs for 72 hours. (G) Western Blots (WB) analyses of MCF7 cell lysates after co-immunoprecipitation (IP) with the indicated antibodies. As negative controls, mouse and rabbit IgG's were used.
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Figure 7: BCL9-2 regulates the transcription of ER in the proximal promoter and interacts with Sp1 in human breast cancer cells(A) Schematic view of regulatory elements within the upstream sequence of the human ESR1 gene that are important for ESR1 gene transcription in breast cancer cells (according to Kos et al., 2001). One previously described G/C-box in promoter B and a G/C-rich element in promoter A as potential Sp1 binding sites are also indicated. The amplified promoter regions by conventional PCR and qRT-PCR following ChIP are indicated below the scheme. (B) Representative examples of the PCR analysis for the indicated promoter fragments. ChIP experiments were performed for the indicated promoter regions of the ESR-1 gene and the GAPDH promoter as control. (C) Re-ChIP experiments of BCL9-2 and Sp1 for the two most proximal promoter regions of the ESR1 gene in MCF7. Representative examples of the PCR analysis following ChIP and re-ChIP with the indicated antibodies and IgG controls are shown. Rabbit IgG (rb IgG) was used as negative control. (D) qRT-PCR analyses for the indicated promoter regions of at least three independent ChIP experiments in MCF7 following immunoprecipitation of the DNA with the indicated antibodies. We performed the absolute quantification using the regression analysis method and calculated the values as percent of input DNA after normalization to the control IgG. (E) qRT-PCR of at least three ChIP experiments following mithramycin A treatment. MCF7 cells were treated with 10 nM mithramycin A or vehicle (0.1% EtOH) for 24 hours prior to fixation. The fold enrichment was calculated using the regression analysis method and normalized to control IgG. (F) Luciferase activity of a reporter containing the most proximal ESR1 gene promoter (WT = wild type), of a G/C box mutant in promoter B (MT-B) and of a G/C-rich element in promoter A (MT-A) as indicated in the scheme. The graph shows a representative dose response experiment performed in triplicates and their range. HEK293 cells were transfected with 100 ng reporter constructs and 0, 12.5, 50 und 100 ng BCL9-2 expression constructs for 72 hours. (G) Western Blots (WB) analyses of MCF7 cell lysates after co-immunoprecipitation (IP) with the indicated antibodies. As negative controls, mouse and rabbit IgG's were used.
Mentions: Since BCL9-2 lacks a conserved DNA binding domain, we included transcription factors that were previously described to bind within the ESR1 gene promoter and activate ER transcription in breast cancer cells (Fig. 7A). First, we analyzed three larger regions in the upstream DNA sequence of the ESR1 gene by conventional PCR following ChIP. The first fragment covered the most proximal promoter A [based on the nomenclature by 33] that is occupied by a complex containing Sp1, p53 and other factors of the basal transcription machinery [7]. Although the binding sites for Sp1 in promoter A have not been described yet, our analyses using Transfac Patch tool [34] or JASPAR database [35] revealed that the ESR1 gene promoter A contains multiple G/C-rich sequences surrounding the transcription start site.

Bottom Line: BCL9-2 overexpression in vivo delayed the mammary involution and induced alveolar hyperplasia.We found, that primary cell cultures of BCL9-2 breast tumors responded to tamoxifen treatment.Finally, we describe a novel mechanism, how BCL9-2 regulates ER transcription by interaction with Sp1 through the proximal ESR1 gene promoter.

View Article: PubMed Central - PubMed

Affiliation: Tumor Biology and Signal Transduction, Georg-August-University Göttingen, Germany. Dept. of Hematology and Medical Oncology, Georg-August-University Göttingen, Germany.

ABSTRACT
The majority of human breast cancers express estrogen receptor alpha (ER), which is important for therapy with anti-estrogens. Here we describe the role of BCL9-2, a proto-oncogene previously characterized as co-activator of Wnt/ß-catenin signaling, for mammary tumorigenesis in mice and human. ER positive human breast cancers showed overexpression of BCL9-2 and tamoxifen treated patients with high BCL9-2 demonstrated a better survival. BCL9-2 was upregulated during puberty and pregnancy in normal mammary epithelia, but downregulated in the involuted gland. BCL9-2 overexpression in vivo delayed the mammary involution and induced alveolar hyperplasia. Moreover, aged BCL9-2 transgenic mice developed ductal-like mammary tumors with high nuclear ER expression. We found, that primary cell cultures of BCL9-2 breast tumors responded to tamoxifen treatment. Moreover, BCL9-2 regulated the expression of ER and the proliferation of human breast cancer cells independently of ß-catenin. Finally, we describe a novel mechanism, how BCL9-2 regulates ER transcription by interaction with Sp1 through the proximal ESR1 gene promoter. In summary, BCL9-2 induces ER positive breast cancers in vivo, regulates ER expression by a novel ß-catenin independent mechanism in breast cancer cells, and might predict the therapy response to tamoxifen treatment.

Show MeSH
Related in: MedlinePlus