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COUP-TFII regulates metastasis of colorectal adenocarcinoma cells by modulating Snail1.

Bao Y, Gu D, Feng W, Sun X, Wang X, Zhang X, Shi Q, Cui G, Yu H, Tang C, Deng A - Br. J. Cancer (2014)

Bottom Line: Overexpression of COUP-TFII was required for cancer cells to metastasise in vivo.Chicken ovalbumin upstream promoter-transcription factor II regulated the transcription and expression of Snail1 by directly targeting the Snail1 promoter and regulated associated genes.Chicken ovalbumin upstream promoter-transcription factor II was found to be a biomarker associated with patient survival and colorectal cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: First Affiliated Hospital, Huzhou Teachers College, the First People's Hospital of Huzhou, Huzhou 313000, China.

ABSTRACT

Background: Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, also known as NR2F2) promotes metastasis by functioning in the tumour microenvironment; however, the role of COUP-TFII in colorectal cancer remains unknown.

Methods: Human colon adenocarcinoma tissues were collected to test COUP-TFII expression. Wound-healing and cell invasion assay were used to evaluate migration and invasion of cells. Chicken ovalbumin upstream promoter-transcription factor II and related protein expression was assessed by immunostaining, immunoblotting and real-time PCR assay. Tamoxifen-inducible COUP-TFII knockout mice were employed to test COUP-TFII functions on colon cancer metastasis in vivo.

Results: Elevated expression of COUP-TFII in colorectal adenocarcinoma tissue correlated with overexpression of the Snail1 transcription factor. High COUP-TFII expression correlated with metastasis and shorter patient survival. Chicken ovalbumin upstream promoter-transcription factor II regulated the migration and invasion of cancer cells. With Snail1, COUP-TFII inhibited expression of adherence molecules such as ZO-1, E-cadherin and β-catenin in colorectal cancer cells. Overexpression of COUP-TFII was required for cancer cells to metastasise in vivo. Chicken ovalbumin upstream promoter-transcription factor II regulated the transcription and expression of Snail1 by directly targeting the Snail1 promoter and regulated associated genes.

Conclusions: Chicken ovalbumin upstream promoter-transcription factor II was crucial for colorectal cancer metastasis and regulated cell migration and metastasis in conjunction with Snail1. Chicken ovalbumin upstream promoter-transcription factor II was found to be a biomarker associated with patient survival and colorectal cancer metastasis.

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Chicken ovalbumin upstream promoter-transcription factor II regulates colon cancer cell invasion by regulating cadherins and MMPs. LOVO cells stably expressing control vector (con), shRNA against COUP-TFII (COUP-TFII KD), shRNA against Snail1 (Snail-1 KD), or COUP-TFII KD + Snail1-overexpressing plasmid were cultured. (A) Expression of F-actin, E-cadherin, β-catenin and ZO-1 by immunofluorescence. Bar, 20 μm. (B) Activity of proMMP2 and proMMP9 in LOVO cell supernatants determined by gelatinase zymography (left). Expression of MMP2 and MMP9 in LOVO cell cytoplasm by western blot (right). (C) Binding to the MMP2 or MMP9 promoter in control (CON) or COUP-TFII knockdown (KD). Cells were transfected with Flag-tagged COUP-TFII. Promoter activity of MMP2 or MMP9 induced by co-expression of COUP-TFII-overexpressing vector. Pull-down assay and ChIP were used to test binding of COUP-TFII and MMP2 or MMP9 promoter. (D) Luciferase activities were measured in LOVO cells cotransfected with COUP-TFII overexpressing or shRNA against COUP-TFII vector. *P<0.01, compared to control. Biotin-labelled oligonucleotides were incubated with nuclear extracts from LOVO cells transiently transfected with COUP-TFII-overexpressing vector. Immobilised streptavidin was used to precipitate the oligonucleotide-transcription factor complexes. Protein–DNA complexes were analysed by immunoblotting using antibodies against FLAG and COUP-TFII.
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fig4: Chicken ovalbumin upstream promoter-transcription factor II regulates colon cancer cell invasion by regulating cadherins and MMPs. LOVO cells stably expressing control vector (con), shRNA against COUP-TFII (COUP-TFII KD), shRNA against Snail1 (Snail-1 KD), or COUP-TFII KD + Snail1-overexpressing plasmid were cultured. (A) Expression of F-actin, E-cadherin, β-catenin and ZO-1 by immunofluorescence. Bar, 20 μm. (B) Activity of proMMP2 and proMMP9 in LOVO cell supernatants determined by gelatinase zymography (left). Expression of MMP2 and MMP9 in LOVO cell cytoplasm by western blot (right). (C) Binding to the MMP2 or MMP9 promoter in control (CON) or COUP-TFII knockdown (KD). Cells were transfected with Flag-tagged COUP-TFII. Promoter activity of MMP2 or MMP9 induced by co-expression of COUP-TFII-overexpressing vector. Pull-down assay and ChIP were used to test binding of COUP-TFII and MMP2 or MMP9 promoter. (D) Luciferase activities were measured in LOVO cells cotransfected with COUP-TFII overexpressing or shRNA against COUP-TFII vector. *P<0.01, compared to control. Biotin-labelled oligonucleotides were incubated with nuclear extracts from LOVO cells transiently transfected with COUP-TFII-overexpressing vector. Immobilised streptavidin was used to precipitate the oligonucleotide-transcription factor complexes. Protein–DNA complexes were analysed by immunoblotting using antibodies against FLAG and COUP-TFII.

Mentions: Given our results on the function of COUP-TFII in metastasis of colon cancer in vivo and in vitro, we hypothesised that COUP-TFII regulated colon cancer cell metastasis through effects on cell–cell contacts. As Figure 4 shows, when confluent LOVO cells were incubated with high concentrations of calcium (1 mM), COUP-TFII or Snail1 knockdown LOVO cells became tightly attached to each other within 12 h, while control LOVO cells did not. In COUP-TFII-deficient or Snail1-deficient LOVO cells, belt-like junctions containing the adherens junction proteins E-cadherin and β-catenin were established 12 h after addition of calcium (Figure 4A). In contrast, only weak signals were detected for E-cadherin and β-catenin in control LOVO cells. Similarly, the tight junction protein ZO-1 was seen to form belt-like structures at the membranes of COUP-TFII-deficient or Snail1-deficient LOVO cells, but only at punctate contacts between control cells (Figure 4A). These potential cell-to-cell junctions between COUP-TFII knockdown cells were not seen in cells transfected with a Snail1-overexpressing plasmid. These results suggested that COUP-TFII regulated colon cancer cell-to-cell junctions through Snail1.


COUP-TFII regulates metastasis of colorectal adenocarcinoma cells by modulating Snail1.

Bao Y, Gu D, Feng W, Sun X, Wang X, Zhang X, Shi Q, Cui G, Yu H, Tang C, Deng A - Br. J. Cancer (2014)

Chicken ovalbumin upstream promoter-transcription factor II regulates colon cancer cell invasion by regulating cadherins and MMPs. LOVO cells stably expressing control vector (con), shRNA against COUP-TFII (COUP-TFII KD), shRNA against Snail1 (Snail-1 KD), or COUP-TFII KD + Snail1-overexpressing plasmid were cultured. (A) Expression of F-actin, E-cadherin, β-catenin and ZO-1 by immunofluorescence. Bar, 20 μm. (B) Activity of proMMP2 and proMMP9 in LOVO cell supernatants determined by gelatinase zymography (left). Expression of MMP2 and MMP9 in LOVO cell cytoplasm by western blot (right). (C) Binding to the MMP2 or MMP9 promoter in control (CON) or COUP-TFII knockdown (KD). Cells were transfected with Flag-tagged COUP-TFII. Promoter activity of MMP2 or MMP9 induced by co-expression of COUP-TFII-overexpressing vector. Pull-down assay and ChIP were used to test binding of COUP-TFII and MMP2 or MMP9 promoter. (D) Luciferase activities were measured in LOVO cells cotransfected with COUP-TFII overexpressing or shRNA against COUP-TFII vector. *P<0.01, compared to control. Biotin-labelled oligonucleotides were incubated with nuclear extracts from LOVO cells transiently transfected with COUP-TFII-overexpressing vector. Immobilised streptavidin was used to precipitate the oligonucleotide-transcription factor complexes. Protein–DNA complexes were analysed by immunoblotting using antibodies against FLAG and COUP-TFII.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Chicken ovalbumin upstream promoter-transcription factor II regulates colon cancer cell invasion by regulating cadherins and MMPs. LOVO cells stably expressing control vector (con), shRNA against COUP-TFII (COUP-TFII KD), shRNA against Snail1 (Snail-1 KD), or COUP-TFII KD + Snail1-overexpressing plasmid were cultured. (A) Expression of F-actin, E-cadherin, β-catenin and ZO-1 by immunofluorescence. Bar, 20 μm. (B) Activity of proMMP2 and proMMP9 in LOVO cell supernatants determined by gelatinase zymography (left). Expression of MMP2 and MMP9 in LOVO cell cytoplasm by western blot (right). (C) Binding to the MMP2 or MMP9 promoter in control (CON) or COUP-TFII knockdown (KD). Cells were transfected with Flag-tagged COUP-TFII. Promoter activity of MMP2 or MMP9 induced by co-expression of COUP-TFII-overexpressing vector. Pull-down assay and ChIP were used to test binding of COUP-TFII and MMP2 or MMP9 promoter. (D) Luciferase activities were measured in LOVO cells cotransfected with COUP-TFII overexpressing or shRNA against COUP-TFII vector. *P<0.01, compared to control. Biotin-labelled oligonucleotides were incubated with nuclear extracts from LOVO cells transiently transfected with COUP-TFII-overexpressing vector. Immobilised streptavidin was used to precipitate the oligonucleotide-transcription factor complexes. Protein–DNA complexes were analysed by immunoblotting using antibodies against FLAG and COUP-TFII.
Mentions: Given our results on the function of COUP-TFII in metastasis of colon cancer in vivo and in vitro, we hypothesised that COUP-TFII regulated colon cancer cell metastasis through effects on cell–cell contacts. As Figure 4 shows, when confluent LOVO cells were incubated with high concentrations of calcium (1 mM), COUP-TFII or Snail1 knockdown LOVO cells became tightly attached to each other within 12 h, while control LOVO cells did not. In COUP-TFII-deficient or Snail1-deficient LOVO cells, belt-like junctions containing the adherens junction proteins E-cadherin and β-catenin were established 12 h after addition of calcium (Figure 4A). In contrast, only weak signals were detected for E-cadherin and β-catenin in control LOVO cells. Similarly, the tight junction protein ZO-1 was seen to form belt-like structures at the membranes of COUP-TFII-deficient or Snail1-deficient LOVO cells, but only at punctate contacts between control cells (Figure 4A). These potential cell-to-cell junctions between COUP-TFII knockdown cells were not seen in cells transfected with a Snail1-overexpressing plasmid. These results suggested that COUP-TFII regulated colon cancer cell-to-cell junctions through Snail1.

Bottom Line: Overexpression of COUP-TFII was required for cancer cells to metastasise in vivo.Chicken ovalbumin upstream promoter-transcription factor II regulated the transcription and expression of Snail1 by directly targeting the Snail1 promoter and regulated associated genes.Chicken ovalbumin upstream promoter-transcription factor II was found to be a biomarker associated with patient survival and colorectal cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: First Affiliated Hospital, Huzhou Teachers College, the First People's Hospital of Huzhou, Huzhou 313000, China.

ABSTRACT

Background: Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, also known as NR2F2) promotes metastasis by functioning in the tumour microenvironment; however, the role of COUP-TFII in colorectal cancer remains unknown.

Methods: Human colon adenocarcinoma tissues were collected to test COUP-TFII expression. Wound-healing and cell invasion assay were used to evaluate migration and invasion of cells. Chicken ovalbumin upstream promoter-transcription factor II and related protein expression was assessed by immunostaining, immunoblotting and real-time PCR assay. Tamoxifen-inducible COUP-TFII knockout mice were employed to test COUP-TFII functions on colon cancer metastasis in vivo.

Results: Elevated expression of COUP-TFII in colorectal adenocarcinoma tissue correlated with overexpression of the Snail1 transcription factor. High COUP-TFII expression correlated with metastasis and shorter patient survival. Chicken ovalbumin upstream promoter-transcription factor II regulated the migration and invasion of cancer cells. With Snail1, COUP-TFII inhibited expression of adherence molecules such as ZO-1, E-cadherin and β-catenin in colorectal cancer cells. Overexpression of COUP-TFII was required for cancer cells to metastasise in vivo. Chicken ovalbumin upstream promoter-transcription factor II regulated the transcription and expression of Snail1 by directly targeting the Snail1 promoter and regulated associated genes.

Conclusions: Chicken ovalbumin upstream promoter-transcription factor II was crucial for colorectal cancer metastasis and regulated cell migration and metastasis in conjunction with Snail1. Chicken ovalbumin upstream promoter-transcription factor II was found to be a biomarker associated with patient survival and colorectal cancer metastasis.

Show MeSH
Related in: MedlinePlus