Limits...
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.

Show MeSH

Related in: MedlinePlus

miR-31 is downregulated in invasive esophageal cancer cells. (A, B) Morphologic, migration and invasive capability of two ESCC cells lines and two EAC cell lines were analyzed by bright field microscopy and Boyden chamber transwell assays. (C) Fold change in expression of 18 miRNAs between the invasive EAC cell line FLO1 and non-invasive cell line OE33. (D-F) Quantitative RT-PCR for relative expression of miR-31. miR-31 expression was normalized to RNU6. (D) High expression of miR-31 in non-invasive ESCC TE11 cells versus invasive TE8 cells. (E) Higher expression in the CP-A cell line compared to CP-B and (F) non-invasive EAC OE33 compared with FLO1. Means ± SD from at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: miR-31 is downregulated in invasive esophageal cancer cells. (A, B) Morphologic, migration and invasive capability of two ESCC cells lines and two EAC cell lines were analyzed by bright field microscopy and Boyden chamber transwell assays. (C) Fold change in expression of 18 miRNAs between the invasive EAC cell line FLO1 and non-invasive cell line OE33. (D-F) Quantitative RT-PCR for relative expression of miR-31. miR-31 expression was normalized to RNU6. (D) High expression of miR-31 in non-invasive ESCC TE11 cells versus invasive TE8 cells. (E) Higher expression in the CP-A cell line compared to CP-B and (F) non-invasive EAC OE33 compared with FLO1. Means ± SD from at least three biological replicates.

Mentions: To investigate the role of miR-31 in esophageal cancers, we examined the expression of miR-31 in ESCC, EAC and Barrett’s esophagus cell lines of differing invasive potential (Figure 1). Comparing esophageal squamous cell carcinoma cell lines, TE11 is less motile than TE8 and displays an epithelial phenotype (Figure 1A). The esophageal adenocarcinoma cell lines OE33 and FLO1 differ in that FLO1 is more mesenchymal and therefore more motile than the OE33 (Figure 1B). After miR-200a and 200b, which are known for their roles in EMT, miR-31 was the most downregulated miRNA in invasive FLO1 cells compared to their less invasive OE33 counterparts by qPCR screen (Figure 1C). Similarly, miR-31 downregulation was observed in TE8 ESCC cell lines compared to TE11 (Figure 1D). Likewise, miR-31 expression was higher in non-invasive cell lines such as the benign Barrett’s esophagus cell line CP-A compared to the metaplastic CP-B cell line (Figure 1E). Furthermore, we confirmed the elevated miR-31 expression in OE33 cells, which have an epithelial phenotype, compared to FLO1 cells (Figure 1F). Next, to focus on the biological significance and regulatory mechanisms of miR-31 expression in invasive adenocarcinoma and squamous cell carcinoma, we expressed miR-31 in invasive ESCC and EAC cell lines and analyzed the effects on cell migration and invasion.Figure 1


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 downregulated in invasive esophageal cancer cells. (A, B) Morphologic, migration and invasive capability of two ESCC cells lines and two EAC cell lines were analyzed by bright field microscopy and Boyden chamber transwell assays. (C) Fold change in expression of 18 miRNAs between the invasive EAC cell line FLO1 and non-invasive cell line OE33. (D-F) Quantitative RT-PCR for relative expression of miR-31. miR-31 expression was normalized to RNU6. (D) High expression of miR-31 in non-invasive ESCC TE11 cells versus invasive TE8 cells. (E) Higher expression in the CP-A cell line compared to CP-B and (F) non-invasive EAC OE33 compared with FLO1. Means ± SD from at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: miR-31 is downregulated in invasive esophageal cancer cells. (A, B) Morphologic, migration and invasive capability of two ESCC cells lines and two EAC cell lines were analyzed by bright field microscopy and Boyden chamber transwell assays. (C) Fold change in expression of 18 miRNAs between the invasive EAC cell line FLO1 and non-invasive cell line OE33. (D-F) Quantitative RT-PCR for relative expression of miR-31. miR-31 expression was normalized to RNU6. (D) High expression of miR-31 in non-invasive ESCC TE11 cells versus invasive TE8 cells. (E) Higher expression in the CP-A cell line compared to CP-B and (F) non-invasive EAC OE33 compared with FLO1. Means ± SD from at least three biological replicates.
Mentions: To investigate the role of miR-31 in esophageal cancers, we examined the expression of miR-31 in ESCC, EAC and Barrett’s esophagus cell lines of differing invasive potential (Figure 1). Comparing esophageal squamous cell carcinoma cell lines, TE11 is less motile than TE8 and displays an epithelial phenotype (Figure 1A). The esophageal adenocarcinoma cell lines OE33 and FLO1 differ in that FLO1 is more mesenchymal and therefore more motile than the OE33 (Figure 1B). After miR-200a and 200b, which are known for their roles in EMT, miR-31 was the most downregulated miRNA in invasive FLO1 cells compared to their less invasive OE33 counterparts by qPCR screen (Figure 1C). Similarly, miR-31 downregulation was observed in TE8 ESCC cell lines compared to TE11 (Figure 1D). Likewise, miR-31 expression was higher in non-invasive cell lines such as the benign Barrett’s esophagus cell line CP-A compared to the metaplastic CP-B cell line (Figure 1E). Furthermore, we confirmed the elevated miR-31 expression in OE33 cells, which have an epithelial phenotype, compared to FLO1 cells (Figure 1F). Next, to focus on the biological significance and regulatory mechanisms of miR-31 expression in invasive adenocarcinoma and squamous cell carcinoma, we expressed miR-31 in invasive ESCC and EAC cell lines and analyzed the effects on cell migration and invasion.Figure 1

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