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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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SOX4, EZH2 and HDAC3 are upregulated and inversely correlate with miR-31 in esophageal cancers. (A) qRT-PCR of SOX4, EZH2, EZH1. HDAC3 and miR-31 in 16 ESCC cell lines. Insert: correlation between mRNA expression of SOX4, EZH2, EZH1, HDAC3 and miR-31 in ESCC cell lines. Statistical analysis was done by ANOVA. (B) SOX4, EZH2, EZH1 and HDAC3 expression in primary esophageal squamous cell carcinomas, ESCC, compared to adjacent normal tissues (Geo dataset GSE20347). (C) SOX4, EZH2, EZH1 and HDAC3 expression in Barrett’s Esophagus lesions and primary esophageal adenocarcinoma tumors, EAC, compared to adjacent normal tissues (Geo dataset GSE13898). (D) Model depicting reciprocal inhibition of miR-31 and SOX4, EZH2 and HDAC3.
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Fig7: SOX4, EZH2 and HDAC3 are upregulated and inversely correlate with miR-31 in esophageal cancers. (A) qRT-PCR of SOX4, EZH2, EZH1. HDAC3 and miR-31 in 16 ESCC cell lines. Insert: correlation between mRNA expression of SOX4, EZH2, EZH1, HDAC3 and miR-31 in ESCC cell lines. Statistical analysis was done by ANOVA. (B) SOX4, EZH2, EZH1 and HDAC3 expression in primary esophageal squamous cell carcinomas, ESCC, compared to adjacent normal tissues (Geo dataset GSE20347). (C) SOX4, EZH2, EZH1 and HDAC3 expression in Barrett’s Esophagus lesions and primary esophageal adenocarcinoma tumors, EAC, compared to adjacent normal tissues (Geo dataset GSE13898). (D) Model depicting reciprocal inhibition of miR-31 and SOX4, EZH2 and HDAC3.

Mentions: To determine the expression of the complex-forming partners, SOX4, EZH2 and HDAC3, in esophageal cancer cell lines and tissues, we used qRT-PCR. We profiled SOX4, EZH2 and HDAC3 in ESCC (Figure 7A) and EAC (data not shown) cell lines and found a strong, inverse correlation between miR-31 and expression of SOX4, EZH2 and HDAC3 in invasive cancers of both histologies. Probing various publicly available datasets (GSE20347, GSE47404, GSE13937) to assess the expression of SOX4, EZH2 and HDAC3 in primary esophageal squamous and adenocarcinoma samples, we found SOX4 and EZH2 to be significantly upregulated in ESCC tumor samples (GSE20347), compared to normal tissues (Figure 7B). In another ESCC dataset (GSE47404), only EZH2 expression (P value = 0.008) was significantly upregulated in metastatic compared to non-metastatic patients. In EAC dataset (GSE13937), we found that SOX4 (P value = 0.003) and HDAC3 (P value = 0.042) were increased in tumor samples compared to adjacent normal tissues (Figure 7C). Taken together, these data identify a molecular circuit where SOX4, EZH2 and HDAC3 target miR-31 to promote esophageal malignancy. Inversely, in non-invasive tumor cells miR-31 targets SOX4, EZH2 and HDAC3 by direct and indirect means to inhibit tumor cell invasion (Figure 7D).Figure 7


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, EZH2 and HDAC3 are upregulated and inversely correlate with miR-31 in esophageal cancers. (A) qRT-PCR of SOX4, EZH2, EZH1. HDAC3 and miR-31 in 16 ESCC cell lines. Insert: correlation between mRNA expression of SOX4, EZH2, EZH1, HDAC3 and miR-31 in ESCC cell lines. Statistical analysis was done by ANOVA. (B) SOX4, EZH2, EZH1 and HDAC3 expression in primary esophageal squamous cell carcinomas, ESCC, compared to adjacent normal tissues (Geo dataset GSE20347). (C) SOX4, EZH2, EZH1 and HDAC3 expression in Barrett’s Esophagus lesions and primary esophageal adenocarcinoma tumors, EAC, compared to adjacent normal tissues (Geo dataset GSE13898). (D) Model depicting reciprocal inhibition of miR-31 and SOX4, EZH2 and HDAC3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: SOX4, EZH2 and HDAC3 are upregulated and inversely correlate with miR-31 in esophageal cancers. (A) qRT-PCR of SOX4, EZH2, EZH1. HDAC3 and miR-31 in 16 ESCC cell lines. Insert: correlation between mRNA expression of SOX4, EZH2, EZH1, HDAC3 and miR-31 in ESCC cell lines. Statistical analysis was done by ANOVA. (B) SOX4, EZH2, EZH1 and HDAC3 expression in primary esophageal squamous cell carcinomas, ESCC, compared to adjacent normal tissues (Geo dataset GSE20347). (C) SOX4, EZH2, EZH1 and HDAC3 expression in Barrett’s Esophagus lesions and primary esophageal adenocarcinoma tumors, EAC, compared to adjacent normal tissues (Geo dataset GSE13898). (D) Model depicting reciprocal inhibition of miR-31 and SOX4, EZH2 and HDAC3.
Mentions: To determine the expression of the complex-forming partners, SOX4, EZH2 and HDAC3, in esophageal cancer cell lines and tissues, we used qRT-PCR. We profiled SOX4, EZH2 and HDAC3 in ESCC (Figure 7A) and EAC (data not shown) cell lines and found a strong, inverse correlation between miR-31 and expression of SOX4, EZH2 and HDAC3 in invasive cancers of both histologies. Probing various publicly available datasets (GSE20347, GSE47404, GSE13937) to assess the expression of SOX4, EZH2 and HDAC3 in primary esophageal squamous and adenocarcinoma samples, we found SOX4 and EZH2 to be significantly upregulated in ESCC tumor samples (GSE20347), compared to normal tissues (Figure 7B). In another ESCC dataset (GSE47404), only EZH2 expression (P value = 0.008) was significantly upregulated in metastatic compared to non-metastatic patients. In EAC dataset (GSE13937), we found that SOX4 (P value = 0.003) and HDAC3 (P value = 0.042) were increased in tumor samples compared to adjacent normal tissues (Figure 7C). Taken together, these data identify a molecular circuit where SOX4, EZH2 and HDAC3 target miR-31 to promote esophageal malignancy. Inversely, in non-invasive tumor cells miR-31 targets SOX4, EZH2 and HDAC3 by direct and indirect means to inhibit tumor cell invasion (Figure 7D).Figure 7

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