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GATA1 induces epithelial-mesenchymal transition in breast cancer cells through PAK5 oncogenic signaling.

Li Y, Ke Q, Shao Y, Zhu G, Li Y, Geng N, Jin F, Li F - Oncotarget (2015)

Bottom Line: Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties.E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT.GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties. E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT. Transcription factor GATA1 has a critical anti-apoptotic role in breast cancer, but its function for metastasis has not been investigated. Here, we found that GATA1, as a novel E-cadherin repressor, promotes EMT in breast cancer cells. GATA1 binds to E-cadherin promoter, down-regulates E-cadherin expression, disrupts intercellular junction and promotes metastasis of breast cancer cell in vivo. Moreover, GATA1 is a new substrate of p21-activated kinase 5 (PAK5), which is phosphorylated on serine 161 and 187 (S161 and S187). GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant. These data indicate that phosphorylated GATA1 recruits more HDAC3/4 to promote transcriptional repression of E-cadherin, leading to the EMT of breast cancer cells. Our findings provide insights into the novel function of GATA1, contributing to a better understanding of the EMT, indicating that GATA1 and its phosphorylation may play an important role in the metastasis of breast cancer.

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GATA1 recruits HDAC3/4 to down-regulate E-cadherin transcription(A) pGL2-E-cad-luc and pRL-TK plasmids were co-transfected with pcDNA-GATA1 or control plasmid into HEK-293 cells and MCF7 cells. Then cells treated with or without TSA for luciferase assay. (B) HEK-293 cells were transfected with pGL2-E-cad-luc plasmid together with HDAC constructs expressing HDAC1–6, respectively. **p < 0.01. (C–D) HEK-293 cells were transfected with pGL2-E-cad-luc, pcDNA-GATA1 and increasing amounts of HDAC3/4 as indicated for Luciferase Assays. Simultaneously, increasing amounts of TSA was added to HEK-293 cells transfected with GATA1 and HDAC3/4 for Luciferase Assays. (E) MCF-7 cells were transfected with Flag-HDAC3/4 expression plasmids. ChIP assay was carried out using anti-GATA1 or anti-Flag antibody, followed by PCR with primers amplifying the E-cadherin promoter region (–1001/–753, –720/–402, –388/–179). ChIP Re-IP, soluble chromatin, prepared from MCF-7 cells transfected with Flag-HDAC3/4, was firstly immunoprecipitated with antibody against GATA1, then reimmunoprecipitated with anti-Flag antibody. (F)In vitro translated Myc-GATA1 or Flag-HDAC3/4 was incubated with GST-HDAC3/4 or GST-GATA1 fusion proteins for GST pull-down assay. (G) HEK-293 cells were transfected with GFP-GATA1 and Flag-HDAC3 or GFP-GATA1 and Flag-HDAC4. Lysates were immunoprecipitated with Flag antibodies and immunoblotted with Flag and GFP antibodies.
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Figure 2: GATA1 recruits HDAC3/4 to down-regulate E-cadherin transcription(A) pGL2-E-cad-luc and pRL-TK plasmids were co-transfected with pcDNA-GATA1 or control plasmid into HEK-293 cells and MCF7 cells. Then cells treated with or without TSA for luciferase assay. (B) HEK-293 cells were transfected with pGL2-E-cad-luc plasmid together with HDAC constructs expressing HDAC1–6, respectively. **p < 0.01. (C–D) HEK-293 cells were transfected with pGL2-E-cad-luc, pcDNA-GATA1 and increasing amounts of HDAC3/4 as indicated for Luciferase Assays. Simultaneously, increasing amounts of TSA was added to HEK-293 cells transfected with GATA1 and HDAC3/4 for Luciferase Assays. (E) MCF-7 cells were transfected with Flag-HDAC3/4 expression plasmids. ChIP assay was carried out using anti-GATA1 or anti-Flag antibody, followed by PCR with primers amplifying the E-cadherin promoter region (–1001/–753, –720/–402, –388/–179). ChIP Re-IP, soluble chromatin, prepared from MCF-7 cells transfected with Flag-HDAC3/4, was firstly immunoprecipitated with antibody against GATA1, then reimmunoprecipitated with anti-Flag antibody. (F)In vitro translated Myc-GATA1 or Flag-HDAC3/4 was incubated with GST-HDAC3/4 or GST-GATA1 fusion proteins for GST pull-down assay. (G) HEK-293 cells were transfected with GFP-GATA1 and Flag-HDAC3 or GFP-GATA1 and Flag-HDAC4. Lysates were immunoprecipitated with Flag antibodies and immunoblotted with Flag and GFP antibodies.

Mentions: Histone deacetylation is one of the best-characterized covalent modifications associated with gene transcriptional repression [23], so we wonder if GATA1 recruits HDACs to down-regulate E-cadherin transcription. The luciferase assays showed that inhibition of HDACs activity by TSA, a known HDACs inhibitor, resulted in the elevation of E-cadherin promoter activity (Figure 2A). Thus, GATA1 down-regulated E-cadherin promoter activity through histone deacetylation. We further tested the effect of six HDACs (HDAC1–6) on E-cadherin transcriptional regulation by GATA1. The luciferase assay results showed that the six HDACs exerted distinct repressive effect on E-cadherin promoter activity, among which HDAC3/4 had a much more prominent effect on E-cadherin repression (Figure 2B). Moreover, HDAC3/4 enhanced the inhibitory effect of GATA1 on E-cadherin promoter activity in a dose-dependent manner and this effect could be dose-dependently reversed by TSA (Figure 2C–2D). Next, the ChIP assay showed that HDAC3/4 bound the same region (–388/–179) of the E-cadherin promoter as GATA1 and the ChIP Re-IP assay indicated that HDAC3/4 and GATA1 acted in a combinatorial fashion on the E-cadherin promoter (Figure 2E). To test whether GATA1 could physically interact with HDAC3/4, in vitro GST-pull down assays were performed and the results indicated that GATA1 bound to HDAC3/4 directly (Figure 2F). In addition, co-immunoprecipitation assays confirmed the interaction of GATA1 with HDAC3/4 in vivo (Figure 2G). Taken together, these results indicate that GATA1 recruits HDAC3/4 to down-regulate E-cadherin expression.


GATA1 induces epithelial-mesenchymal transition in breast cancer cells through PAK5 oncogenic signaling.

Li Y, Ke Q, Shao Y, Zhu G, Li Y, Geng N, Jin F, Li F - Oncotarget (2015)

GATA1 recruits HDAC3/4 to down-regulate E-cadherin transcription(A) pGL2-E-cad-luc and pRL-TK plasmids were co-transfected with pcDNA-GATA1 or control plasmid into HEK-293 cells and MCF7 cells. Then cells treated with or without TSA for luciferase assay. (B) HEK-293 cells were transfected with pGL2-E-cad-luc plasmid together with HDAC constructs expressing HDAC1–6, respectively. **p < 0.01. (C–D) HEK-293 cells were transfected with pGL2-E-cad-luc, pcDNA-GATA1 and increasing amounts of HDAC3/4 as indicated for Luciferase Assays. Simultaneously, increasing amounts of TSA was added to HEK-293 cells transfected with GATA1 and HDAC3/4 for Luciferase Assays. (E) MCF-7 cells were transfected with Flag-HDAC3/4 expression plasmids. ChIP assay was carried out using anti-GATA1 or anti-Flag antibody, followed by PCR with primers amplifying the E-cadherin promoter region (–1001/–753, –720/–402, –388/–179). ChIP Re-IP, soluble chromatin, prepared from MCF-7 cells transfected with Flag-HDAC3/4, was firstly immunoprecipitated with antibody against GATA1, then reimmunoprecipitated with anti-Flag antibody. (F)In vitro translated Myc-GATA1 or Flag-HDAC3/4 was incubated with GST-HDAC3/4 or GST-GATA1 fusion proteins for GST pull-down assay. (G) HEK-293 cells were transfected with GFP-GATA1 and Flag-HDAC3 or GFP-GATA1 and Flag-HDAC4. Lysates were immunoprecipitated with Flag antibodies and immunoblotted with Flag and GFP antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: GATA1 recruits HDAC3/4 to down-regulate E-cadherin transcription(A) pGL2-E-cad-luc and pRL-TK plasmids were co-transfected with pcDNA-GATA1 or control plasmid into HEK-293 cells and MCF7 cells. Then cells treated with or without TSA for luciferase assay. (B) HEK-293 cells were transfected with pGL2-E-cad-luc plasmid together with HDAC constructs expressing HDAC1–6, respectively. **p < 0.01. (C–D) HEK-293 cells were transfected with pGL2-E-cad-luc, pcDNA-GATA1 and increasing amounts of HDAC3/4 as indicated for Luciferase Assays. Simultaneously, increasing amounts of TSA was added to HEK-293 cells transfected with GATA1 and HDAC3/4 for Luciferase Assays. (E) MCF-7 cells were transfected with Flag-HDAC3/4 expression plasmids. ChIP assay was carried out using anti-GATA1 or anti-Flag antibody, followed by PCR with primers amplifying the E-cadherin promoter region (–1001/–753, –720/–402, –388/–179). ChIP Re-IP, soluble chromatin, prepared from MCF-7 cells transfected with Flag-HDAC3/4, was firstly immunoprecipitated with antibody against GATA1, then reimmunoprecipitated with anti-Flag antibody. (F)In vitro translated Myc-GATA1 or Flag-HDAC3/4 was incubated with GST-HDAC3/4 or GST-GATA1 fusion proteins for GST pull-down assay. (G) HEK-293 cells were transfected with GFP-GATA1 and Flag-HDAC3 or GFP-GATA1 and Flag-HDAC4. Lysates were immunoprecipitated with Flag antibodies and immunoblotted with Flag and GFP antibodies.
Mentions: Histone deacetylation is one of the best-characterized covalent modifications associated with gene transcriptional repression [23], so we wonder if GATA1 recruits HDACs to down-regulate E-cadherin transcription. The luciferase assays showed that inhibition of HDACs activity by TSA, a known HDACs inhibitor, resulted in the elevation of E-cadherin promoter activity (Figure 2A). Thus, GATA1 down-regulated E-cadherin promoter activity through histone deacetylation. We further tested the effect of six HDACs (HDAC1–6) on E-cadherin transcriptional regulation by GATA1. The luciferase assay results showed that the six HDACs exerted distinct repressive effect on E-cadherin promoter activity, among which HDAC3/4 had a much more prominent effect on E-cadherin repression (Figure 2B). Moreover, HDAC3/4 enhanced the inhibitory effect of GATA1 on E-cadherin promoter activity in a dose-dependent manner and this effect could be dose-dependently reversed by TSA (Figure 2C–2D). Next, the ChIP assay showed that HDAC3/4 bound the same region (–388/–179) of the E-cadherin promoter as GATA1 and the ChIP Re-IP assay indicated that HDAC3/4 and GATA1 acted in a combinatorial fashion on the E-cadherin promoter (Figure 2E). To test whether GATA1 could physically interact with HDAC3/4, in vitro GST-pull down assays were performed and the results indicated that GATA1 bound to HDAC3/4 directly (Figure 2F). In addition, co-immunoprecipitation assays confirmed the interaction of GATA1 with HDAC3/4 in vivo (Figure 2G). Taken together, these results indicate that GATA1 recruits HDAC3/4 to down-regulate E-cadherin expression.

Bottom Line: Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties.E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT.GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties. E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT. Transcription factor GATA1 has a critical anti-apoptotic role in breast cancer, but its function for metastasis has not been investigated. Here, we found that GATA1, as a novel E-cadherin repressor, promotes EMT in breast cancer cells. GATA1 binds to E-cadherin promoter, down-regulates E-cadherin expression, disrupts intercellular junction and promotes metastasis of breast cancer cell in vivo. Moreover, GATA1 is a new substrate of p21-activated kinase 5 (PAK5), which is phosphorylated on serine 161 and 187 (S161 and S187). GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant. These data indicate that phosphorylated GATA1 recruits more HDAC3/4 to promote transcriptional repression of E-cadherin, leading to the EMT of breast cancer cells. Our findings provide insights into the novel function of GATA1, contributing to a better understanding of the EMT, indicating that GATA1 and its phosphorylation may play an important role in the metastasis of breast cancer.

Show MeSH
Related in: MedlinePlus