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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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ChIP analysis of ERα with the vav1 promoter DNA.(A) Schematic representation of the vav1 proximal promoter region. The predicted transcription factors and hERE sites were framed by boxes. Horizontal arrows indicated the primers used for PCR in ChIP assays. TSS: transcription start site. hERE: half estrogen response element (ERE). (B) T47D cells were treated with E2 (10−7 mol/L) or DMSO (solvent control) for 4 h and ChIP analysis was performed with anti-ERα antibody or control IgG. Two sets of primers specific for +59 to +340 region containing hERE sites (upper panel) or the −232 to +71 region of the vav1 promoter (lower panel) were used in PCR. The PCR products were detected by agarose gel electrophoresis. The input represented the DNA in crude cell extract before the immunoprecipitation. (C) T47D Cells were treated with the reagents as indicated in the left side, and ChIP assay were carried on using primers specific for −232 to +71 of vav1 promoter. The PCR products were resolved by agarose gel electrophoresis. The bar chart beside each example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “**” 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-g004: ChIP analysis of ERα with the vav1 promoter DNA.(A) Schematic representation of the vav1 proximal promoter region. The predicted transcription factors and hERE sites were framed by boxes. Horizontal arrows indicated the primers used for PCR in ChIP assays. TSS: transcription start site. hERE: half estrogen response element (ERE). (B) T47D cells were treated with E2 (10−7 mol/L) or DMSO (solvent control) for 4 h and ChIP analysis was performed with anti-ERα antibody or control IgG. Two sets of primers specific for +59 to +340 region containing hERE sites (upper panel) or the −232 to +71 region of the vav1 promoter (lower panel) were used in PCR. The PCR products were detected by agarose gel electrophoresis. The input represented the DNA in crude cell extract before the immunoprecipitation. (C) T47D Cells were treated with the reagents as indicated in the left side, and ChIP assay were carried on using primers specific for −232 to +71 of vav1 promoter. The PCR products were resolved by agarose gel electrophoresis. The bar chart beside each example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “**” indicates P<0.01 versus DMSO treatment and “a**” indicates P<0.01 versus E2 treatment by unpaired student T test.

Mentions: The involvement of ERα in estrogen-induced vav1 transcription led us to examine the vav1 proximal promoter for conserved ERE sequence in silico by rVista2.0 (http://rvista.dcode.org/) and TRANSFAC (http://www.cbrc.jp/htbin/nph-tfsearch). However, the search result revealed no perfect ERE at the vav1 promoter region, rather, there were two half-ERE sites (hERE) located at the positions +165 to +169 bp and +273 to +277 bp to TSS, respectively (Fig. 4A). As previously reported, ERE-like sequence, such as two half ERE sites, can bind with estrogen activated ER even though they were separated by hundreds of base pairs [9][38]. Thus we set to verify if ERα bound to the hERE sites at vav1 promoter by ChIP analysis. The primers corresponding to the region spanning the two hERE sites (+59 to +340) were designed accordingly. As shown in Figure 4B upper panel, the sample prior to immunoprecipitation (Input) exhibited a positive hERE region, whereas was detected negative in the post-immunoprecipitated sample (ERα), indicating that ERα did not interact with the hERE sites. Unexpectedly, the region −232 to +71 was found in association with ERα (Fig. 4B, lower panel, third lane from the left), though there was no consensus binding site for ER. Furthermore the recruitment of ERα was increased by ∼1.7 fold upon E2 treatment (Fig. 4B, lower panel, sixth lane from the left, P<0.01), and reduced by Tamoxifen treatment (Fig. 4C, P<0.01 versus DMSO and E2 treatment). The above results demonstrated that ERα was involved in the transcriptional activation of vav1 gene by association with the promoter region other than the hERE sites, implying an indirect binding of ERα to the promoter region, perhaps through other transcription factors.


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)

ChIP analysis of ERα with the vav1 promoter DNA.(A) Schematic representation of the vav1 proximal promoter region. The predicted transcription factors and hERE sites were framed by boxes. Horizontal arrows indicated the primers used for PCR in ChIP assays. TSS: transcription start site. hERE: half estrogen response element (ERE). (B) T47D cells were treated with E2 (10−7 mol/L) or DMSO (solvent control) for 4 h and ChIP analysis was performed with anti-ERα antibody or control IgG. Two sets of primers specific for +59 to +340 region containing hERE sites (upper panel) or the −232 to +71 region of the vav1 promoter (lower panel) were used in PCR. The PCR products were detected by agarose gel electrophoresis. The input represented the DNA in crude cell extract before the immunoprecipitation. (C) T47D Cells were treated with the reagents as indicated in the left side, and ChIP assay were carried on using primers specific for −232 to +71 of vav1 promoter. The PCR products were resolved by agarose gel electrophoresis. The bar chart beside each example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “**” 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-g004: ChIP analysis of ERα with the vav1 promoter DNA.(A) Schematic representation of the vav1 proximal promoter region. The predicted transcription factors and hERE sites were framed by boxes. Horizontal arrows indicated the primers used for PCR in ChIP assays. TSS: transcription start site. hERE: half estrogen response element (ERE). (B) T47D cells were treated with E2 (10−7 mol/L) or DMSO (solvent control) for 4 h and ChIP analysis was performed with anti-ERα antibody or control IgG. Two sets of primers specific for +59 to +340 region containing hERE sites (upper panel) or the −232 to +71 region of the vav1 promoter (lower panel) were used in PCR. The PCR products were detected by agarose gel electrophoresis. The input represented the DNA in crude cell extract before the immunoprecipitation. (C) T47D Cells were treated with the reagents as indicated in the left side, and ChIP assay were carried on using primers specific for −232 to +71 of vav1 promoter. The PCR products were resolved by agarose gel electrophoresis. The bar chart beside each example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “**” indicates P<0.01 versus DMSO treatment and “a**” indicates P<0.01 versus E2 treatment by unpaired student T test.
Mentions: The involvement of ERα in estrogen-induced vav1 transcription led us to examine the vav1 proximal promoter for conserved ERE sequence in silico by rVista2.0 (http://rvista.dcode.org/) and TRANSFAC (http://www.cbrc.jp/htbin/nph-tfsearch). However, the search result revealed no perfect ERE at the vav1 promoter region, rather, there were two half-ERE sites (hERE) located at the positions +165 to +169 bp and +273 to +277 bp to TSS, respectively (Fig. 4A). As previously reported, ERE-like sequence, such as two half ERE sites, can bind with estrogen activated ER even though they were separated by hundreds of base pairs [9][38]. Thus we set to verify if ERα bound to the hERE sites at vav1 promoter by ChIP analysis. The primers corresponding to the region spanning the two hERE sites (+59 to +340) were designed accordingly. As shown in Figure 4B upper panel, the sample prior to immunoprecipitation (Input) exhibited a positive hERE region, whereas was detected negative in the post-immunoprecipitated sample (ERα), indicating that ERα did not interact with the hERE sites. Unexpectedly, the region −232 to +71 was found in association with ERα (Fig. 4B, lower panel, third lane from the left), though there was no consensus binding site for ER. Furthermore the recruitment of ERα was increased by ∼1.7 fold upon E2 treatment (Fig. 4B, lower panel, sixth lane from the left, P<0.01), and reduced by Tamoxifen treatment (Fig. 4C, P<0.01 versus DMSO and E2 treatment). The above results demonstrated that ERα was involved in the transcriptional activation of vav1 gene by association with the promoter region other than the hERE sites, implying an indirect binding of ERα to the promoter region, perhaps through other transcription factors.

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