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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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ER-mediated Vav1 expression.(A) MCF7 and T47D cells were treated with E2 (10−7 mol/L) or DMSO for 24 h. The relative level of vav1 mRNA was determined by qRT-PCR and was presented by the ratio of vav1 mRNA of E2-treated samples to that of DMSO-treated control samples and presented as y-axis. The data represented the mean value±S.D. of three independent experiments. (B and C) MCF7 and T47D cells were exposed to E2 (10-7 mol/L) for 0 to 72 h (B), or increasing concentration of E2 for 48 h (C). (D and E) MCF7 and T47D cells were pre-treated with ICI 182,780 (4×10−7 mol/L) (D) or increasing concentration of Tamoxifen for 30 min (E) before adding E2 (10−7 mol/L) for 48 h. The DMSO treatment was used as a solvent control. The Vav1 expression in above treated samples was analyzed by Western Blot with anti-Vav1 antibody, with tubulin as protein loading control. The bar chart below each example blot represents the normalized protein level of Vav1 to Tubulin of three independent experiments. The DMSO treatment was set as 1 to indicate the basal level of Vav1 expression. “**” indicates P<0.01 versus DMSO treatment and “a**” indicates P<0.01 versus E2 treatment by unpaired student T test.
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pone-0099052-g002: ER-mediated Vav1 expression.(A) MCF7 and T47D cells were treated with E2 (10−7 mol/L) or DMSO for 24 h. The relative level of vav1 mRNA was determined by qRT-PCR and was presented by the ratio of vav1 mRNA of E2-treated samples to that of DMSO-treated control samples and presented as y-axis. The data represented the mean value±S.D. of three independent experiments. (B and C) MCF7 and T47D cells were exposed to E2 (10-7 mol/L) for 0 to 72 h (B), or increasing concentration of E2 for 48 h (C). (D and E) MCF7 and T47D cells were pre-treated with ICI 182,780 (4×10−7 mol/L) (D) or increasing concentration of Tamoxifen for 30 min (E) before adding E2 (10−7 mol/L) for 48 h. The DMSO treatment was used as a solvent control. The Vav1 expression in above treated samples was analyzed by Western Blot with anti-Vav1 antibody, with tubulin as protein loading control. The bar chart below each example blot represents the normalized protein level of Vav1 to Tubulin of three independent experiments. The DMSO treatment was set as 1 to indicate the basal level of Vav1 expression. “**” indicates P<0.01 versus DMSO treatment and “a**” indicates P<0.01 versus E2 treatment by unpaired student T test.

Mentions: A correlation has been observed between the progression of breast cancer and the exposure to estrogen, which modulates the transcription of many genes by binding and activating ERs [2]–[5]. From the results of tissue immunohistochemistry [27] and cell lines Western blot (Fig. 1), higher Vav1 expression was visualized in ER positive breast tumors or cells than that in ER negative samples or cells. We speculated that estrogen-ER was involved in the control of vav1 gene expression. Two ER-positive cell lines, MCF7 and T47D, were tested for Vav1 expression in the presence or absence of 17β-estradiol (E2). After treatment with 10−7 mol/L of E2 or DMSO as control, the mRNA transcript of vav1 was measured by qRT-PCR. As shown in Figure 2A, E2 induced an increase in vav1 mRNA expression by 3.15-fold in MCF7 and 2.86-fold in T47D in reference to the DMSO control (P<0.01), suggesting that E2 enhanced the transcription 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)

ER-mediated Vav1 expression.(A) MCF7 and T47D cells were treated with E2 (10−7 mol/L) or DMSO for 24 h. The relative level of vav1 mRNA was determined by qRT-PCR and was presented by the ratio of vav1 mRNA of E2-treated samples to that of DMSO-treated control samples and presented as y-axis. The data represented the mean value±S.D. of three independent experiments. (B and C) MCF7 and T47D cells were exposed to E2 (10-7 mol/L) for 0 to 72 h (B), or increasing concentration of E2 for 48 h (C). (D and E) MCF7 and T47D cells were pre-treated with ICI 182,780 (4×10−7 mol/L) (D) or increasing concentration of Tamoxifen for 30 min (E) before adding E2 (10−7 mol/L) for 48 h. The DMSO treatment was used as a solvent control. The Vav1 expression in above treated samples was analyzed by Western Blot with anti-Vav1 antibody, with tubulin as protein loading control. The bar chart below each example blot represents the normalized protein level of Vav1 to Tubulin of three independent experiments. The DMSO treatment was set as 1 to indicate the basal level of Vav1 expression. “**” indicates P<0.01 versus DMSO treatment and “a**” indicates P<0.01 versus E2 treatment by unpaired student T test.
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Related In: Results  -  Collection

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pone-0099052-g002: ER-mediated Vav1 expression.(A) MCF7 and T47D cells were treated with E2 (10−7 mol/L) or DMSO for 24 h. The relative level of vav1 mRNA was determined by qRT-PCR and was presented by the ratio of vav1 mRNA of E2-treated samples to that of DMSO-treated control samples and presented as y-axis. The data represented the mean value±S.D. of three independent experiments. (B and C) MCF7 and T47D cells were exposed to E2 (10-7 mol/L) for 0 to 72 h (B), or increasing concentration of E2 for 48 h (C). (D and E) MCF7 and T47D cells were pre-treated with ICI 182,780 (4×10−7 mol/L) (D) or increasing concentration of Tamoxifen for 30 min (E) before adding E2 (10−7 mol/L) for 48 h. The DMSO treatment was used as a solvent control. The Vav1 expression in above treated samples was analyzed by Western Blot with anti-Vav1 antibody, with tubulin as protein loading control. The bar chart below each example blot represents the normalized protein level of Vav1 to Tubulin of three independent experiments. The DMSO treatment was set as 1 to indicate the basal level of Vav1 expression. “**” indicates P<0.01 versus DMSO treatment and “a**” indicates P<0.01 versus E2 treatment by unpaired student T test.
Mentions: A correlation has been observed between the progression of breast cancer and the exposure to estrogen, which modulates the transcription of many genes by binding and activating ERs [2]–[5]. From the results of tissue immunohistochemistry [27] and cell lines Western blot (Fig. 1), higher Vav1 expression was visualized in ER positive breast tumors or cells than that in ER negative samples or cells. We speculated that estrogen-ER was involved in the control of vav1 gene expression. Two ER-positive cell lines, MCF7 and T47D, were tested for Vav1 expression in the presence or absence of 17β-estradiol (E2). After treatment with 10−7 mol/L of E2 or DMSO as control, the mRNA transcript of vav1 was measured by qRT-PCR. As shown in Figure 2A, E2 induced an increase in vav1 mRNA expression by 3.15-fold in MCF7 and 2.86-fold in T47D in reference to the DMSO control (P<0.01), suggesting that E2 enhanced the transcription 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