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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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Effect of Vav1 in cell cycle progression.(A) T47D cells were infected with lentivirus particles that expressed Vav1-specific shRNA or shRNA with a scramble sequence (served as a control). The homogenous cells were first synchronized to G0/G1 phase and then treated with DMSO as control or E2 (10−7 mol/L) for 36 h before harvest. The expression of Vav1 and Cyclin D1 were analyzed by Western Blot with indicated antibodies respectively. The density of the bands was quantitated by Quantity One software (Bio-Rad, version 4.4.0, CA, USA). The bar chart represents the normalized protein level of Cyclin D1 to tubulin of three independent experiments. “**” indicates P<0.01 versus DMSO treatment of Control, and “a**” indicates P<0.01 versus E2 treatment of Control by unpaired student T test. (B) Another aliquots of above cells were stained by PI and analyzed by flow cytometer for DNA contents. The decrease in the percentage of cells in G0/G1-phase before and after the treatment by DMSO or E2 was calculated and plotted as y-axis and labeled on the bar graph. The differences between two T47D-Ctrl and T47D-ShVav1 cells were also marked. The data represented the mean ±S.D. of three independent experiments. “**” indicates P<0.01 versus corresponding control group by unpaired student T test.
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pone-0099052-g006: Effect of Vav1 in cell cycle progression.(A) T47D cells were infected with lentivirus particles that expressed Vav1-specific shRNA or shRNA with a scramble sequence (served as a control). The homogenous cells were first synchronized to G0/G1 phase and then treated with DMSO as control or E2 (10−7 mol/L) for 36 h before harvest. The expression of Vav1 and Cyclin D1 were analyzed by Western Blot with indicated antibodies respectively. The density of the bands was quantitated by Quantity One software (Bio-Rad, version 4.4.0, CA, USA). The bar chart represents the normalized protein level of Cyclin D1 to tubulin of three independent experiments. “**” indicates P<0.01 versus DMSO treatment of Control, and “a**” indicates P<0.01 versus E2 treatment of Control by unpaired student T test. (B) Another aliquots of above cells were stained by PI and analyzed by flow cytometer for DNA contents. The decrease in the percentage of cells in G0/G1-phase before and after the treatment by DMSO or E2 was calculated and plotted as y-axis and labeled on the bar graph. The differences between two T47D-Ctrl and T47D-ShVav1 cells were also marked. The data represented the mean ±S.D. of three independent experiments. “**” indicates P<0.01 versus corresponding control group by unpaired student T test.

Mentions: E2 is identified as a causative factor of breast cancers and well-characterized to induce cell growth in ER-positive breast tumors [34]. As Vav1 is also involved in cell proliferation in lung cancer and pancreatic cancer cells [22], [23], we speculated that Vav1 participated in cell cycle progression of breast cancer cells under the control of E2. Stable cell lines, T47D-ShVav1 expressing short hairpin RNA for Vav1, and T47D-Ctrl expressing a scrambled sequence, were established by lentivirus-based transduction. The expression of Vav1 in these cell lines was verified as shown in Figure 6A, and the level of Vav1 expressed in shVav1 cells was reduced (Fig. 6A, top panel, the left two lanes). The E2 treatment elevated the Vav1 expression proportionally (Fig. 6A, top panel, the right two lanes). The expression of Cyclin D1 was also determined as a commonly recognized factor for cell cycle progression (Fig. 6A, middle panel). In the absence of E2, shVav1 reduced Cyclin D1 by 50% (P<0.01) in comparison with the control (left lane). E2 induced a significant 2.31-fold increase of Cyclin D1 (Fig. 6A, third lane from the left, P<0.01) in coordination with Vav1, and that was reduced by the shRNA knockdown of Vav1 (P<0.01 versus E2 treatment of Control).


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)

Effect of Vav1 in cell cycle progression.(A) T47D cells were infected with lentivirus particles that expressed Vav1-specific shRNA or shRNA with a scramble sequence (served as a control). The homogenous cells were first synchronized to G0/G1 phase and then treated with DMSO as control or E2 (10−7 mol/L) for 36 h before harvest. The expression of Vav1 and Cyclin D1 were analyzed by Western Blot with indicated antibodies respectively. The density of the bands was quantitated by Quantity One software (Bio-Rad, version 4.4.0, CA, USA). The bar chart represents the normalized protein level of Cyclin D1 to tubulin of three independent experiments. “**” indicates P<0.01 versus DMSO treatment of Control, and “a**” indicates P<0.01 versus E2 treatment of Control by unpaired student T test. (B) Another aliquots of above cells were stained by PI and analyzed by flow cytometer for DNA contents. The decrease in the percentage of cells in G0/G1-phase before and after the treatment by DMSO or E2 was calculated and plotted as y-axis and labeled on the bar graph. The differences between two T47D-Ctrl and T47D-ShVav1 cells were also marked. The data represented the mean ±S.D. of three independent experiments. “**” indicates P<0.01 versus corresponding control group by unpaired student T test.
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Related In: Results  -  Collection

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pone-0099052-g006: Effect of Vav1 in cell cycle progression.(A) T47D cells were infected with lentivirus particles that expressed Vav1-specific shRNA or shRNA with a scramble sequence (served as a control). The homogenous cells were first synchronized to G0/G1 phase and then treated with DMSO as control or E2 (10−7 mol/L) for 36 h before harvest. The expression of Vav1 and Cyclin D1 were analyzed by Western Blot with indicated antibodies respectively. The density of the bands was quantitated by Quantity One software (Bio-Rad, version 4.4.0, CA, USA). The bar chart represents the normalized protein level of Cyclin D1 to tubulin of three independent experiments. “**” indicates P<0.01 versus DMSO treatment of Control, and “a**” indicates P<0.01 versus E2 treatment of Control by unpaired student T test. (B) Another aliquots of above cells were stained by PI and analyzed by flow cytometer for DNA contents. The decrease in the percentage of cells in G0/G1-phase before and after the treatment by DMSO or E2 was calculated and plotted as y-axis and labeled on the bar graph. The differences between two T47D-Ctrl and T47D-ShVav1 cells were also marked. The data represented the mean ±S.D. of three independent experiments. “**” indicates P<0.01 versus corresponding control group by unpaired student T test.
Mentions: E2 is identified as a causative factor of breast cancers and well-characterized to induce cell growth in ER-positive breast tumors [34]. As Vav1 is also involved in cell proliferation in lung cancer and pancreatic cancer cells [22], [23], we speculated that Vav1 participated in cell cycle progression of breast cancer cells under the control of E2. Stable cell lines, T47D-ShVav1 expressing short hairpin RNA for Vav1, and T47D-Ctrl expressing a scrambled sequence, were established by lentivirus-based transduction. The expression of Vav1 in these cell lines was verified as shown in Figure 6A, and the level of Vav1 expressed in shVav1 cells was reduced (Fig. 6A, top panel, the left two lanes). The E2 treatment elevated the Vav1 expression proportionally (Fig. 6A, top panel, the right two lanes). The expression of Cyclin D1 was also determined as a commonly recognized factor for cell cycle progression (Fig. 6A, middle panel). In the absence of E2, shVav1 reduced Cyclin D1 by 50% (P<0.01) in comparison with the control (left lane). E2 induced a significant 2.31-fold increase of Cyclin D1 (Fig. 6A, third lane from the left, P<0.01) in coordination with Vav1, and that was reduced by the shRNA knockdown of Vav1 (P<0.01 versus E2 treatment of Control).

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