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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 knockdown suppresses migration, invasion and growth of invasive esophageal cancer cells. TE8 and FLO1 cells were transfected with non-targeting shRNA control or shSOX4. (A) SOX4 protein expression was analyzed by Western blotting. (B, C) miR-31 expression in response to SOX4 knockdown was measured by qRT-PCR, miR-191 and miR-423-5p were used as controls. miR-31, miR-191, miR-423-5p expression was normalized to RNU6. (D, E) Cell migration was measured using Boyden chamber transwell assays 24 hour post-transfection. (F, G) Invasion was measured by Marigel-coated transwell assays 24 hour post-transfection. (H, I) Cell viability was evaluated using the WST-1 assay. Results are means ± SD from at least three biological replicates.
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Fig5: SOX4 knockdown suppresses migration, invasion and growth of invasive esophageal cancer cells. TE8 and FLO1 cells were transfected with non-targeting shRNA control or shSOX4. (A) SOX4 protein expression was analyzed by Western blotting. (B, C) miR-31 expression in response to SOX4 knockdown was measured by qRT-PCR, miR-191 and miR-423-5p were used as controls. miR-31, miR-191, miR-423-5p expression was normalized to RNU6. (D, E) Cell migration was measured using Boyden chamber transwell assays 24 hour post-transfection. (F, G) Invasion was measured by Marigel-coated transwell assays 24 hour post-transfection. (H, I) Cell viability was evaluated using the WST-1 assay. Results are means ± SD from at least three biological replicates.

Mentions: Based on the observation that miR-31 targets both SOX4 and EZH2 and given that SOX4 binds to the EZH2 promoter to activate its transcription, we examined whether SOX4 leads to feed forward activation of EZH2, and subsequent miR-31 silencing. TE8 and FLO1 cells, which express high levels of SOX4 and EZH2, showed the lowest expression level of miR-31. SOX4 knockdown (Figure 5A) using shRNA led to significant upregulation of miR-31 in TE8 and FLO1 cells, while miR-191 and miR-423-5p, used as controls, did not show any significant change (Figure 5B and C). We next investigated if loss of SOX4 functionally mimics overexpression of miR-31 in esophageal cancer cells. To assess the role of SOX4, we studied the migratory and invasive potential of TE8 and FLO1 cells following SOX4 knockdown with shRNA. Suppression of SOX4 inhibited transwell migration (Figure 5D and E) and invasion through Matrigel-coated Boyden chambers (Figure 5F and G) in TE8 and FLO1 cells. SOX4 knockdown cells also showed a significant reduction in proliferation (Figure 5H and I). Taken together, these data implicate SOX4 as a key mediator of the tumor-suppressive effects of miR-31 in this system.Figure 5


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 knockdown suppresses migration, invasion and growth of invasive esophageal cancer cells. TE8 and FLO1 cells were transfected with non-targeting shRNA control or shSOX4. (A) SOX4 protein expression was analyzed by Western blotting. (B, C) miR-31 expression in response to SOX4 knockdown was measured by qRT-PCR, miR-191 and miR-423-5p were used as controls. miR-31, miR-191, miR-423-5p expression was normalized to RNU6. (D, E) Cell migration was measured using Boyden chamber transwell assays 24 hour post-transfection. (F, G) Invasion was measured by Marigel-coated transwell assays 24 hour post-transfection. (H, I) Cell viability was evaluated using the WST-1 assay. Results are 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

Fig5: SOX4 knockdown suppresses migration, invasion and growth of invasive esophageal cancer cells. TE8 and FLO1 cells were transfected with non-targeting shRNA control or shSOX4. (A) SOX4 protein expression was analyzed by Western blotting. (B, C) miR-31 expression in response to SOX4 knockdown was measured by qRT-PCR, miR-191 and miR-423-5p were used as controls. miR-31, miR-191, miR-423-5p expression was normalized to RNU6. (D, E) Cell migration was measured using Boyden chamber transwell assays 24 hour post-transfection. (F, G) Invasion was measured by Marigel-coated transwell assays 24 hour post-transfection. (H, I) Cell viability was evaluated using the WST-1 assay. Results are means ± SD from at least three biological replicates.
Mentions: Based on the observation that miR-31 targets both SOX4 and EZH2 and given that SOX4 binds to the EZH2 promoter to activate its transcription, we examined whether SOX4 leads to feed forward activation of EZH2, and subsequent miR-31 silencing. TE8 and FLO1 cells, which express high levels of SOX4 and EZH2, showed the lowest expression level of miR-31. SOX4 knockdown (Figure 5A) using shRNA led to significant upregulation of miR-31 in TE8 and FLO1 cells, while miR-191 and miR-423-5p, used as controls, did not show any significant change (Figure 5B and C). We next investigated if loss of SOX4 functionally mimics overexpression of miR-31 in esophageal cancer cells. To assess the role of SOX4, we studied the migratory and invasive potential of TE8 and FLO1 cells following SOX4 knockdown with shRNA. Suppression of SOX4 inhibited transwell migration (Figure 5D and E) and invasion through Matrigel-coated Boyden chambers (Figure 5F and G) in TE8 and FLO1 cells. SOX4 knockdown cells also showed a significant reduction in proliferation (Figure 5H and I). Taken together, these data implicate SOX4 as a key mediator of the tumor-suppressive effects of miR-31 in this system.Figure 5

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