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SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells.

Koumangoye RB, Andl T, Taubenslag KJ, Zilberman ST, Taylor CJ, Loomans HA, Andl CD - Mol. Cancer (2015)

Bottom Line: We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR.Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, 2213 Garland Ave. 10445 MRB IV, Nashville, TN, 37232-6840, USA. rainelli.koumangoye@vanderbilt.edu.

ABSTRACT

Background: Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear.

Methods: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.

Results: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.

Conclusions: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

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Related in: MedlinePlus

miR-31 is epigenetically repressed in invasive esophageal cancer cells. TE8 and FLO1 cells were treated at indicated concentrations with Polycomb/EZH2 inhibitor (DZNep), Histone deacetylase inhibitor (SAHA) and DNA methyl-transferase inhibitors (AZA). (A) DZNep treatment for 72 hours dose-dependently inhibits EZH2 and EZH1 in FLO1 and TE8 cells as shown by western blotting. (B) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with DZNep for 72 hours. (C) SAHA treatment for 48 hours inhibits HDAC3, EZH2 and EZH1 in TE8 and FLO1 cells in a dose-dependent manner. (D) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with SAHA for 48 hours. (E-F) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with 5-aza-deoxycytidine (AZA) for 72 hours. miR-31 expression was normalized to RNU6. Results are means ± SD from at least three biological replicates, ANOVA was used for statistical analysis.
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Fig3: miR-31 is epigenetically repressed in invasive esophageal cancer cells. TE8 and FLO1 cells were treated at indicated concentrations with Polycomb/EZH2 inhibitor (DZNep), Histone deacetylase inhibitor (SAHA) and DNA methyl-transferase inhibitors (AZA). (A) DZNep treatment for 72 hours dose-dependently inhibits EZH2 and EZH1 in FLO1 and TE8 cells as shown by western blotting. (B) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with DZNep for 72 hours. (C) SAHA treatment for 48 hours inhibits HDAC3, EZH2 and EZH1 in TE8 and FLO1 cells in a dose-dependent manner. (D) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with SAHA for 48 hours. (E-F) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with 5-aza-deoxycytidine (AZA) for 72 hours. miR-31 expression was normalized to RNU6. Results are means ± SD from at least three biological replicates, ANOVA was used for statistical analysis.

Mentions: Prior studies report that miR-31 expression is epigenetically silenced through promoter hypermethylation at CpG islands, as well as polycomb-mediated histone methylation [8,11]. We therefore speculated that loss of miR-31 in invasive esophageal cancer cells could be mediated, in part, by DNA and histone methylation. To determine the effect of PRC2 on miR-31 expression, we utilized the PRC2 inhibitor, 3-deazaneplanocin (DZNep). DZNep treatment of TE8 and FLO1 cells resulted in a decrease in EZH2 expression and caused a dose-dependent increase in miR-31 expression in both of our invasive cell lines (Figure 3A, B, respectively). DZNep treatment also led to a decrease in SOX4 protein level in FLO1 cells (Figure 3A). SOX4 was recently shown to upregulate EZH2 expression [30]. Using Western blot and qRT-PCR, we evaluated the effect of a pan-HDAC inhibitor (SAHA) on miR-31 expression. We found that SAHA led to a decrease in HDAC3, EZH2 and EZH1 protein levels in TE8 and FLO1 cells (Figure 3C). Most importantly, miR-31 expression was significantly upregulated in both cell lines following SAHA treatment (Figure 3D). To test whether promoter methylation at CpG islands was involved in miR-31 silencing, we used the DNA methylation inhibitor 5′AZA-Deoxy-Cytidine (AZA). Treatment with AZA significantly increased the expression of miR-31 in TE8 cells and to a lesser extent in FLO1 cells (Figure 3E and F). These data suggest that PRC2, HDAC and DNA methylation are involved in miR-31 epigenetic silencing.Figure 3


SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells.

Koumangoye RB, Andl T, Taubenslag KJ, Zilberman ST, Taylor CJ, Loomans HA, Andl CD - Mol. Cancer (2015)

miR-31 is epigenetically repressed in invasive esophageal cancer cells. TE8 and FLO1 cells were treated at indicated concentrations with Polycomb/EZH2 inhibitor (DZNep), Histone deacetylase inhibitor (SAHA) and DNA methyl-transferase inhibitors (AZA). (A) DZNep treatment for 72 hours dose-dependently inhibits EZH2 and EZH1 in FLO1 and TE8 cells as shown by western blotting. (B) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with DZNep for 72 hours. (C) SAHA treatment for 48 hours inhibits HDAC3, EZH2 and EZH1 in TE8 and FLO1 cells in a dose-dependent manner. (D) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with SAHA for 48 hours. (E-F) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with 5-aza-deoxycytidine (AZA) for 72 hours. miR-31 expression was normalized to RNU6. Results are means ± SD from at least three biological replicates, ANOVA was used for statistical analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: miR-31 is epigenetically repressed in invasive esophageal cancer cells. TE8 and FLO1 cells were treated at indicated concentrations with Polycomb/EZH2 inhibitor (DZNep), Histone deacetylase inhibitor (SAHA) and DNA methyl-transferase inhibitors (AZA). (A) DZNep treatment for 72 hours dose-dependently inhibits EZH2 and EZH1 in FLO1 and TE8 cells as shown by western blotting. (B) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with DZNep for 72 hours. (C) SAHA treatment for 48 hours inhibits HDAC3, EZH2 and EZH1 in TE8 and FLO1 cells in a dose-dependent manner. (D) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with SAHA for 48 hours. (E-F) qRT-PCR analysis of miR-31 expression in TE8 and FLO1 cells treated with 5-aza-deoxycytidine (AZA) for 72 hours. miR-31 expression was normalized to RNU6. Results are means ± SD from at least three biological replicates, ANOVA was used for statistical analysis.
Mentions: Prior studies report that miR-31 expression is epigenetically silenced through promoter hypermethylation at CpG islands, as well as polycomb-mediated histone methylation [8,11]. We therefore speculated that loss of miR-31 in invasive esophageal cancer cells could be mediated, in part, by DNA and histone methylation. To determine the effect of PRC2 on miR-31 expression, we utilized the PRC2 inhibitor, 3-deazaneplanocin (DZNep). DZNep treatment of TE8 and FLO1 cells resulted in a decrease in EZH2 expression and caused a dose-dependent increase in miR-31 expression in both of our invasive cell lines (Figure 3A, B, respectively). DZNep treatment also led to a decrease in SOX4 protein level in FLO1 cells (Figure 3A). SOX4 was recently shown to upregulate EZH2 expression [30]. Using Western blot and qRT-PCR, we evaluated the effect of a pan-HDAC inhibitor (SAHA) on miR-31 expression. We found that SAHA led to a decrease in HDAC3, EZH2 and EZH1 protein levels in TE8 and FLO1 cells (Figure 3C). Most importantly, miR-31 expression was significantly upregulated in both cell lines following SAHA treatment (Figure 3D). To test whether promoter methylation at CpG islands was involved in miR-31 silencing, we used the DNA methylation inhibitor 5′AZA-Deoxy-Cytidine (AZA). Treatment with AZA significantly increased the expression of miR-31 in TE8 cells and to a lesser extent in FLO1 cells (Figure 3E and F). These data suggest that PRC2, HDAC and DNA methylation are involved in miR-31 epigenetic silencing.Figure 3

Bottom Line: We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR.Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, 2213 Garland Ave. 10445 MRB IV, Nashville, TN, 37232-6840, USA. rainelli.koumangoye@vanderbilt.edu.

ABSTRACT

Background: Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear.

Methods: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.

Results: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.

Conclusions: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

Show MeSH
Related in: MedlinePlus