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Estrogen induces Vav1 expression in human breast cancer cells.

Du MJ, Chen XD, Zhou XL, Wan YJ, Lan B, Zhang CZ, Cao Y - PLoS ONE (2014)

Bottom Line: Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter.Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle.The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin, P. R. China.

ABSTRACT
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17β-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not β. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

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Activation of vav1 promoter by ERα.MCF7 cells were transfected with the vav1 luciferase reporter gene, and cultured in RPMI 1640 medium for 24 h. The cells were pre-treated with increasing concentration of Tamoxifen (A) or ICI 182 782 at 4×10−7 mol/L (B) for 30 min, followed by E2 treatment for another 48 h. Or the cells were treated with E2 (10−7 mol/L), PPT (10−7 mol/L), or DPN (10−7 mol/L) for 48 h (C). The DMSO treatment served as solvent control. The induction fold of luciferase activity was measured as described in Methods, and plotted as the ratio to that of the control in y-axis. The data represented the mean±S.D. of three independent experiments. “**” and “N.S.” indicate P<0.01 and P>0.05, respectively, versus DMSO treatment by unpaired student T test; “a**” and “aN.S.” indicate P<0.01 and P>0.05, respectively, versus E2 treatment.
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pone-0099052-g003: Activation of vav1 promoter by ERα.MCF7 cells were transfected with the vav1 luciferase reporter gene, and cultured in RPMI 1640 medium for 24 h. The cells were pre-treated with increasing concentration of Tamoxifen (A) or ICI 182 782 at 4×10−7 mol/L (B) for 30 min, followed by E2 treatment for another 48 h. Or the cells were treated with E2 (10−7 mol/L), PPT (10−7 mol/L), or DPN (10−7 mol/L) for 48 h (C). The DMSO treatment served as solvent control. The induction fold of luciferase activity was measured as described in Methods, and plotted as the ratio to that of the control in y-axis. The data represented the mean±S.D. of three independent experiments. “**” and “N.S.” indicate P<0.01 and P>0.05, respectively, versus DMSO treatment by unpaired student T test; “a**” and “aN.S.” indicate P<0.01 and P>0.05, respectively, versus E2 treatment.

Mentions: As E2-ER efficiently enhanced Vav1 protein as well as mRNA expression, we predicted that ERs would function as a transcriptional activator for vav1 gene promoter. The minimal regulatory sequences of vav1 proximal promoter region, which covered nucleotide (nt) −287 to +301 relative to Transcription Start Site (TSS), was constructed in plasmid pGL3 as described [30], and the resulting plasmid was named pVav1-Luc. In agreement with Vav1 protein expression in Figure 2, E2 treatment induced a maximal activation of vav1 promoter, nearly 3 fold above the control (DMSO treatment) (Fig. 3A, P<0.01). The presence of Tamoxifen decreased the vav1 promoter activity to the basal level, and the promoter activity exhibited a negative correlation with the increasing concentration of the drug (Fig. 3A). Similarly, ICI 182,780 suppressed the reporter gene by 1.33 fold (Fig. 3B, P<0.01 versus E2 treatment). The above results suggested that ERs were involved in the activation of vav1 promoter activity, and thus the transcriptional activation of vav1 gene.


Estrogen induces Vav1 expression in human breast cancer cells.

Du MJ, Chen XD, Zhou XL, Wan YJ, Lan B, Zhang CZ, Cao Y - PLoS ONE (2014)

Activation of vav1 promoter by ERα.MCF7 cells were transfected with the vav1 luciferase reporter gene, and cultured in RPMI 1640 medium for 24 h. The cells were pre-treated with increasing concentration of Tamoxifen (A) or ICI 182 782 at 4×10−7 mol/L (B) for 30 min, followed by E2 treatment for another 48 h. Or the cells were treated with E2 (10−7 mol/L), PPT (10−7 mol/L), or DPN (10−7 mol/L) for 48 h (C). The DMSO treatment served as solvent control. The induction fold of luciferase activity was measured as described in Methods, and plotted as the ratio to that of the control in y-axis. The data represented the mean±S.D. of three independent experiments. “**” and “N.S.” indicate P<0.01 and P>0.05, respectively, versus DMSO treatment by unpaired student T test; “a**” and “aN.S.” indicate P<0.01 and P>0.05, respectively, versus E2 treatment.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048212&req=5

pone-0099052-g003: Activation of vav1 promoter by ERα.MCF7 cells were transfected with the vav1 luciferase reporter gene, and cultured in RPMI 1640 medium for 24 h. The cells were pre-treated with increasing concentration of Tamoxifen (A) or ICI 182 782 at 4×10−7 mol/L (B) for 30 min, followed by E2 treatment for another 48 h. Or the cells were treated with E2 (10−7 mol/L), PPT (10−7 mol/L), or DPN (10−7 mol/L) for 48 h (C). The DMSO treatment served as solvent control. The induction fold of luciferase activity was measured as described in Methods, and plotted as the ratio to that of the control in y-axis. The data represented the mean±S.D. of three independent experiments. “**” and “N.S.” indicate P<0.01 and P>0.05, respectively, versus DMSO treatment by unpaired student T test; “a**” and “aN.S.” indicate P<0.01 and P>0.05, respectively, versus E2 treatment.
Mentions: As E2-ER efficiently enhanced Vav1 protein as well as mRNA expression, we predicted that ERs would function as a transcriptional activator for vav1 gene promoter. The minimal regulatory sequences of vav1 proximal promoter region, which covered nucleotide (nt) −287 to +301 relative to Transcription Start Site (TSS), was constructed in plasmid pGL3 as described [30], and the resulting plasmid was named pVav1-Luc. In agreement with Vav1 protein expression in Figure 2, E2 treatment induced a maximal activation of vav1 promoter, nearly 3 fold above the control (DMSO treatment) (Fig. 3A, P<0.01). The presence of Tamoxifen decreased the vav1 promoter activity to the basal level, and the promoter activity exhibited a negative correlation with the increasing concentration of the drug (Fig. 3A). Similarly, ICI 182,780 suppressed the reporter gene by 1.33 fold (Fig. 3B, P<0.01 versus E2 treatment). The above results suggested that ERs were involved in the activation of vav1 promoter activity, and thus the transcriptional activation of vav1 gene.

Bottom Line: Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter.Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle.The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin, P. R. China.

ABSTRACT
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17β-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not β. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

Show MeSH
Related in: MedlinePlus