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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 directly targets SOX4 and indirectly targets EZH2 and HDAC3. (A) Computational analysis revealed one miR-31 binding site in the 3′UTR of SOX4. The upper panel shows the region containing the miR-31 binding site. The mutated SOX4 3′UTR seed region is indicated. A SOX4 3′UTR fragment containing wild type (WT OLIGO) or mutant (MUT OLIGO) of the miR-31-binding sequence was cloned into the downstream of the luciferase reporter gene. The lower panel shows the nucleotide sequence alignment of the predicted miR-31 binding site in the 3′UTR of SOX4 of six species. (B) HEK293, TE8 and FLO1 cells were co-transfected with psiCHECK-2 dual Renilla/Firefly luciferase plasmid containing either wild-type, wild-type oligo or mutant oligo of SOX4 3′UTR (indicated as WT, WT OLIGO and MUT OLIGO) with either pBABE empty vector control or pBABE-miR-31 vector. Luciferase activity was determined 48 hrs after transfection. (C) TE8 and FLO1 cells were transfected with miR-31 vector or empty vector control and cell lysates were analyzed after 72 hrs for SOX4, EZH2, EZH1 and HDAC3 by western blotting. α-tubulin was used as an internal control. (D) qRT-PCR analysis of SOX4, EZH2, EZH1 and HDAC expression in TE8 and FLO1 cells transfected with miR-31 or empty vector control. (E, F) qRT-PCR analysis of TE11 cells transfected with SOX4 or empty vector control. (G, H) qRT-PCR analysis of TE11 cells transfected with EZH2 or empty vector control. miR-31 expression was normalized to RNU6 and SOX4, EZH2, EZH1 and HDAC3 were normalized to GAPDH. Results are means ± SD from at least three biological replicates.
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Fig4: miR-31 directly targets SOX4 and indirectly targets EZH2 and HDAC3. (A) Computational analysis revealed one miR-31 binding site in the 3′UTR of SOX4. The upper panel shows the region containing the miR-31 binding site. The mutated SOX4 3′UTR seed region is indicated. A SOX4 3′UTR fragment containing wild type (WT OLIGO) or mutant (MUT OLIGO) of the miR-31-binding sequence was cloned into the downstream of the luciferase reporter gene. The lower panel shows the nucleotide sequence alignment of the predicted miR-31 binding site in the 3′UTR of SOX4 of six species. (B) HEK293, TE8 and FLO1 cells were co-transfected with psiCHECK-2 dual Renilla/Firefly luciferase plasmid containing either wild-type, wild-type oligo or mutant oligo of SOX4 3′UTR (indicated as WT, WT OLIGO and MUT OLIGO) with either pBABE empty vector control or pBABE-miR-31 vector. Luciferase activity was determined 48 hrs after transfection. (C) TE8 and FLO1 cells were transfected with miR-31 vector or empty vector control and cell lysates were analyzed after 72 hrs for SOX4, EZH2, EZH1 and HDAC3 by western blotting. α-tubulin was used as an internal control. (D) qRT-PCR analysis of SOX4, EZH2, EZH1 and HDAC expression in TE8 and FLO1 cells transfected with miR-31 or empty vector control. (E, F) qRT-PCR analysis of TE11 cells transfected with SOX4 or empty vector control. (G, H) qRT-PCR analysis of TE11 cells transfected with EZH2 or empty vector control. miR-31 expression was normalized to RNU6 and SOX4, EZH2, EZH1 and HDAC3 were normalized to GAPDH. Results are means ± SD from at least three biological replicates.

Mentions: A study by Asangani et al. recently showed that genetic and epigenetic loss of miR-31 leads to a feed forward upregulation of EZH2 [11]. However, no mechanism was proposed. Previously, EZH2 was reported to interact with HDAC3 to repress miR-29 in lymphomas [37]. More recent work shows that SOX4 binds to the EZH2 promoter, thereby upregulating EZH2 expression [30]. We hypothesized that SOX4 initiates the feed forward activation of EZH2, which in turn represses miR-31. Analysis of the SOX4 3′-UTR using microrna.org (maintained at cBio, the Computational Biology Center at Memorial Sloan-Kettering Cancer Center) predicted a miR-31 binding site (Figure 4A). A sequence alignment search showed that the miR-31 target sequence in the SOX4 3′-UTR is conserved in humans and most great apes (Figure 4A). To test whether SOX4 is regulated by miR-31 through direct binding to its 3′UTR, we used psiCHECK2 SOX4 full length 3′-UTR plasmid (WT) [3], and constructed two derivatives, SOX4 WT 3′-UTR oligo plasmid (WT OLIGO) and SOX4 mutant 3′-UTR oligo plasmid (MUT OLIGO) (Figure 4A). The WT OLIGO plasmid contained a 71-nucleotide region including the miR-31 target sequence. In the SOX4 mutant 3′-UTR (MUT OLIGO), 4 nucleotides in the seed sequence were mutated [3]. When co-transfected into HEK-293 cells, the luciferase reporter, SOX4 WT 3′-UTR and miR-31 plasmid showed reduced luciferase activity compared to co-transfection with miR-31 empty control vector (Figure 4B). This suppressive effect was reversed by the four-nucleotide substitution in the miR-31 binding sequence. Similarly, the suppressive effect of miR-31 on the SOX4 3′-UTR activity was observed in the esophageal tumor cell lines, TE8 and FLO1 (Figure 4B). In line with these results, overexpression of miR-31 in FLO1 cells suppressed the expression of SOX4 at both the protein (Figure 4C) and mRNA level (Figure 4D). As previously reported [11], our data confirm that miR-31 inhibits EZH2 expression (Figure 4C and D) whereas EZH1 expression was unchanged. Interestingly, miR-31 decreased HDAC3 on protein (Figure 4C) and mRNA levels (Figure 4D). However, target prediction algorithms do not detect any putative binding site for miR-31 in the 5′UTR, 3′UTR or coding sequence of HDAC3. Taken together, these results demonstrate that SOX4 is a direct target of miR-31, while EZH2 and HDAC3 are indirect targets.Figure 4


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 directly targets SOX4 and indirectly targets EZH2 and HDAC3. (A) Computational analysis revealed one miR-31 binding site in the 3′UTR of SOX4. The upper panel shows the region containing the miR-31 binding site. The mutated SOX4 3′UTR seed region is indicated. A SOX4 3′UTR fragment containing wild type (WT OLIGO) or mutant (MUT OLIGO) of the miR-31-binding sequence was cloned into the downstream of the luciferase reporter gene. The lower panel shows the nucleotide sequence alignment of the predicted miR-31 binding site in the 3′UTR of SOX4 of six species. (B) HEK293, TE8 and FLO1 cells were co-transfected with psiCHECK-2 dual Renilla/Firefly luciferase plasmid containing either wild-type, wild-type oligo or mutant oligo of SOX4 3′UTR (indicated as WT, WT OLIGO and MUT OLIGO) with either pBABE empty vector control or pBABE-miR-31 vector. Luciferase activity was determined 48 hrs after transfection. (C) TE8 and FLO1 cells were transfected with miR-31 vector or empty vector control and cell lysates were analyzed after 72 hrs for SOX4, EZH2, EZH1 and HDAC3 by western blotting. α-tubulin was used as an internal control. (D) qRT-PCR analysis of SOX4, EZH2, EZH1 and HDAC expression in TE8 and FLO1 cells transfected with miR-31 or empty vector control. (E, F) qRT-PCR analysis of TE11 cells transfected with SOX4 or empty vector control. (G, H) qRT-PCR analysis of TE11 cells transfected with EZH2 or empty vector control. miR-31 expression was normalized to RNU6 and SOX4, EZH2, EZH1 and HDAC3 were normalized to GAPDH. Results are means ± SD from at least three biological replicates.
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Related In: Results  -  Collection

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Fig4: miR-31 directly targets SOX4 and indirectly targets EZH2 and HDAC3. (A) Computational analysis revealed one miR-31 binding site in the 3′UTR of SOX4. The upper panel shows the region containing the miR-31 binding site. The mutated SOX4 3′UTR seed region is indicated. A SOX4 3′UTR fragment containing wild type (WT OLIGO) or mutant (MUT OLIGO) of the miR-31-binding sequence was cloned into the downstream of the luciferase reporter gene. The lower panel shows the nucleotide sequence alignment of the predicted miR-31 binding site in the 3′UTR of SOX4 of six species. (B) HEK293, TE8 and FLO1 cells were co-transfected with psiCHECK-2 dual Renilla/Firefly luciferase plasmid containing either wild-type, wild-type oligo or mutant oligo of SOX4 3′UTR (indicated as WT, WT OLIGO and MUT OLIGO) with either pBABE empty vector control or pBABE-miR-31 vector. Luciferase activity was determined 48 hrs after transfection. (C) TE8 and FLO1 cells were transfected with miR-31 vector or empty vector control and cell lysates were analyzed after 72 hrs for SOX4, EZH2, EZH1 and HDAC3 by western blotting. α-tubulin was used as an internal control. (D) qRT-PCR analysis of SOX4, EZH2, EZH1 and HDAC expression in TE8 and FLO1 cells transfected with miR-31 or empty vector control. (E, F) qRT-PCR analysis of TE11 cells transfected with SOX4 or empty vector control. (G, H) qRT-PCR analysis of TE11 cells transfected with EZH2 or empty vector control. miR-31 expression was normalized to RNU6 and SOX4, EZH2, EZH1 and HDAC3 were normalized to GAPDH. Results are means ± SD from at least three biological replicates.
Mentions: A study by Asangani et al. recently showed that genetic and epigenetic loss of miR-31 leads to a feed forward upregulation of EZH2 [11]. However, no mechanism was proposed. Previously, EZH2 was reported to interact with HDAC3 to repress miR-29 in lymphomas [37]. More recent work shows that SOX4 binds to the EZH2 promoter, thereby upregulating EZH2 expression [30]. We hypothesized that SOX4 initiates the feed forward activation of EZH2, which in turn represses miR-31. Analysis of the SOX4 3′-UTR using microrna.org (maintained at cBio, the Computational Biology Center at Memorial Sloan-Kettering Cancer Center) predicted a miR-31 binding site (Figure 4A). A sequence alignment search showed that the miR-31 target sequence in the SOX4 3′-UTR is conserved in humans and most great apes (Figure 4A). To test whether SOX4 is regulated by miR-31 through direct binding to its 3′UTR, we used psiCHECK2 SOX4 full length 3′-UTR plasmid (WT) [3], and constructed two derivatives, SOX4 WT 3′-UTR oligo plasmid (WT OLIGO) and SOX4 mutant 3′-UTR oligo plasmid (MUT OLIGO) (Figure 4A). The WT OLIGO plasmid contained a 71-nucleotide region including the miR-31 target sequence. In the SOX4 mutant 3′-UTR (MUT OLIGO), 4 nucleotides in the seed sequence were mutated [3]. When co-transfected into HEK-293 cells, the luciferase reporter, SOX4 WT 3′-UTR and miR-31 plasmid showed reduced luciferase activity compared to co-transfection with miR-31 empty control vector (Figure 4B). This suppressive effect was reversed by the four-nucleotide substitution in the miR-31 binding sequence. Similarly, the suppressive effect of miR-31 on the SOX4 3′-UTR activity was observed in the esophageal tumor cell lines, TE8 and FLO1 (Figure 4B). In line with these results, overexpression of miR-31 in FLO1 cells suppressed the expression of SOX4 at both the protein (Figure 4C) and mRNA level (Figure 4D). As previously reported [11], our data confirm that miR-31 inhibits EZH2 expression (Figure 4C and D) whereas EZH1 expression was unchanged. Interestingly, miR-31 decreased HDAC3 on protein (Figure 4C) and mRNA levels (Figure 4D). However, target prediction algorithms do not detect any putative binding site for miR-31 in the 5′UTR, 3′UTR or coding sequence of HDAC3. Taken together, these results demonstrate that SOX4 is a direct target of miR-31, while EZH2 and HDAC3 are indirect targets.Figure 4

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