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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

SOX4 interacts with EZH2 and HDAC3. (A) TE8 cell lysates were immunoprecipitated with SOX4, EZH2, HDAC3 or IgG control antibodies and immunoblots were probed for indicated antibodies. (B) FLO1 cell lysates were pulled-down with SOX4, EZH2, HDAC3 antibody or IgG control and immunoblots were probed for indicated antibodies. (C) Quantitative ChIP assay showing H3K27me3, and HDAC3 enrichment on the proximal and distal promoters regions of miR-31 and its host genes MIR31HG.
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Fig6: SOX4 interacts with EZH2 and HDAC3. (A) TE8 cell lysates were immunoprecipitated with SOX4, EZH2, HDAC3 or IgG control antibodies and immunoblots were probed for indicated antibodies. (B) FLO1 cell lysates were pulled-down with SOX4, EZH2, HDAC3 antibody or IgG control and immunoblots were probed for indicated antibodies. (C) Quantitative ChIP assay showing H3K27me3, and HDAC3 enrichment on the proximal and distal promoters regions of miR-31 and its host genes MIR31HG.

Mentions: PRC2 is known to recruit DNA methyl-transferases (DNMTs), HDACs and other chromatin modifying enzymes to repress the transcription of developmental genes [43]. Because SOX4 was recently shown to interact with other transcription factors, we tested whether SOX4 forms a co-repressor complex with EZH2 and HDAC3 to silence miR-31 expression. Using anti-SOX4 antibody for co-immunoprecipitation, cell lysates from TE8 (Figure 6A) and FLO1 cells (Figure 6B) showed interactions between SOX4 and EZH2, HDAC3 and H3K27me3 (Figure 6A, B). We next performed co-immunoprecipitation assays with anti-EZH2 antibody and showed that EZH2 equally interacts with SOX4 (Figure 6A, B). Immunoprecipitates obtained with EZH2 antibody detected also HDAC3, H3K27me3, and Suz12 (Figure 6A, B). To test whether HDAC3 interacts with SOX4 and EZH2, HDAC3-antibody was used for pull down. Lysates immunoprecipitated with HDAC3-specific antibody contained EZH2, SOX4, H3K27me3 in TE8 cells (Figure 6A), but only a faint band of EZH2 in FLO1 cells (Figure 6B). Suz12 was not detected after pull-down with HDAC3-specific antibody in either cell line.Figure 6


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)

SOX4 interacts with EZH2 and HDAC3. (A) TE8 cell lysates were immunoprecipitated with SOX4, EZH2, HDAC3 or IgG control antibodies and immunoblots were probed for indicated antibodies. (B) FLO1 cell lysates were pulled-down with SOX4, EZH2, HDAC3 antibody or IgG control and immunoblots were probed for indicated antibodies. (C) Quantitative ChIP assay showing H3K27me3, and HDAC3 enrichment on the proximal and distal promoters regions of miR-31 and its host genes MIR31HG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374188&req=5

Fig6: SOX4 interacts with EZH2 and HDAC3. (A) TE8 cell lysates were immunoprecipitated with SOX4, EZH2, HDAC3 or IgG control antibodies and immunoblots were probed for indicated antibodies. (B) FLO1 cell lysates were pulled-down with SOX4, EZH2, HDAC3 antibody or IgG control and immunoblots were probed for indicated antibodies. (C) Quantitative ChIP assay showing H3K27me3, and HDAC3 enrichment on the proximal and distal promoters regions of miR-31 and its host genes MIR31HG.
Mentions: PRC2 is known to recruit DNA methyl-transferases (DNMTs), HDACs and other chromatin modifying enzymes to repress the transcription of developmental genes [43]. Because SOX4 was recently shown to interact with other transcription factors, we tested whether SOX4 forms a co-repressor complex with EZH2 and HDAC3 to silence miR-31 expression. Using anti-SOX4 antibody for co-immunoprecipitation, cell lysates from TE8 (Figure 6A) and FLO1 cells (Figure 6B) showed interactions between SOX4 and EZH2, HDAC3 and H3K27me3 (Figure 6A, B). We next performed co-immunoprecipitation assays with anti-EZH2 antibody and showed that EZH2 equally interacts with SOX4 (Figure 6A, B). Immunoprecipitates obtained with EZH2 antibody detected also HDAC3, H3K27me3, and Suz12 (Figure 6A, B). To test whether HDAC3 interacts with SOX4 and EZH2, HDAC3-antibody was used for pull down. Lysates immunoprecipitated with HDAC3-specific antibody contained EZH2, SOX4, H3K27me3 in TE8 cells (Figure 6A), but only a faint band of EZH2 in FLO1 cells (Figure 6B). Suz12 was not detected after pull-down with HDAC3-specific antibody in either cell line.Figure 6

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