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

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.

Figure 1

Chicken ovalbumin upstream promoter-transcription factor II and Snail1 are overexpressed in colon adenocarcinoma tissue. (A) Expression of COUP-TFII and Snail-1 in human colorectal adenocarcinoma tissue. Western blots were used to determine expression of COUP-TFII and Snail1 in human colorectal adenocarcinoma tissue. Lanes contain equal amounts of nuclear protein from colorectal adenocarcinoma tissue (T) and paired normal tissue (N). (B) Quantification of image density from A. *P<0.01 compared to normal. (C) Representive images were shown for the expression of COUP-TFII and Snail1 in 326 human colorectal adenocarcinoma tissue samples, determined by western blot. (D) Survival curves for colorectal adenocarcinoma patients with either high or low COUP-TFII (P<0.01). (E) Human colon adenocarcinoma LOVO or HT29 cells stably expressing control vector (CON), shRNA against COUP-TFII (COUP-TFII KD), shRNA against Snail1 (Snail-1 KD), COUP-TFII KD + Snail-1 overexpressing (Snail-1 OV), COUP-TFII overexpressing (COUP-TFII OV), or COUP-TFII OV + Snail-1 KD were cultured. Expression of COUP-TFII and Snail1 by western blot. Tubulin was the loading control. Density of bands were quantified.

Figure 2

Chicken ovalbumin upstream promoter-transcription factor II regulation of colon cancer cell invasion is dependent on Snail1. Human colon adenocarcinoma LOVO or HT29 cells were transfected and cultured as described in Figure 1E. (A and B) Wound-healing assay with LOVO or HT29 cells transfected with indicated vectors showing wound width and percent closure of the original wound from triplicate plates. *P<0.01 compared to the same time points for control non-transfected (CON) wounded LOVO or HT29 cells. (C and D) LOVO or HT29 cells transfected with indicated vectors that penetrated Matrigel-coated filters. Quantitation is the mean number of cells in 10 random microscope fields. Data are mean±s.d., n=3; *P<0.01.

Figure 3

Chicken ovalbumin upstream promoter-transcription factor II regulates metastasis of colon cancer in vivo in mice. Wild-type (WT) or COUP-TFII^−/− mice were used to generate DMH/DSS-induced colon cancer models. (A) Typical appearance of metastatic foci on control livers (arrows). (B) Number of metastatic foci from A were calculated. *P<0.01 compared to WT. (C) Haematoxylin and eosin (H&E) staining for liver tissue. The arrow shows the tumour. Bar, 200 μm. (D) Immunohistochemistry was used to evaluate expression of E-cadherin and β-catenin from collected colon adenocarcinoma tissues. Bar, 50 μm. (E) Western blot showing expression of MMP2 and MMP9 in tumours from wild-type mice (WT) or COUP-TFII^−/− mice (COUP-TFII^−/−). (F) Control LOVO cells (CON) or COUP-TFII-depleted LOVO cells (COUP-TFII KD) were injected into spleens of nude mice. After 30 days, mice were killed and metastatic foci (arrows) on livers were examined. (G) Number of metastatic foci from F was calculated. *P<0.01 compared to CON. (H) H&E staining for liver tissue. Bar, 200 μm.

Figure 4

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.

Figure 5

Overexpression of COUP-TFII in intestinal epithelial cells results in tumour characteristics. (A) Expression of COUP-TFII and Snail-1 in HIEC, LOVO and HT29 cells by western blot. (B) HIEC cells were transfected with a COUP-TFII-overexpressing plasmid (COUP-TFII), and cytoplasmic COUP-TFII and Snail-1 were examined by western blot. (C) Wound-healing assay of control and COUP-TFII-overexpressing HIEC cells showing wound width for percent closure of the original wound in triplicate plates. Similar results were obtained in three experiments. (D) Migrated control and COUP-TFII-overexpressing cells that penetrated Matrigel-coated filters. (E) Snail-1 mRNA in HIEC, LOVO and HT29 cells was quantified by real-time PCR (*P< 0.01 compared to HIEC). (F) Snail-1 mRNA in control and COUP-TFII-overexpressing cells quantified by real-time PCR (*P<0.01 compared to CON).

Figure 6

Chicken ovalbumin upstream promoter-transcription factor II positively regulates Snail-1 expression through direct transcriptional activation. (A and B) Extracts of LOVO cells stably expressing control vector (CON), shRNA against COUP-TFII (COUP-TFII KD), or COUP-TFII-overexpressing vector (COUP-TFII OV) were examined by chromatin immunoprecipitation with anti-COUP-TFII antibody or non-immune IgG and real-time PCR of Snail-1 promoter regions containing or lacking high-affinity COUP-TFII-binding sites (black and white boxes in the map). Supershift assay was used by using antibody against COUP-TFII. −, control without antibody; antibody, with antibody against COUP-TFII (C) EMSA using LOVO cells stably expressing control vector (CON), COUP-TFII shRNA (COUP-TFII KD), or COUP-TFII-overexpressing vector (COUP-TFII OV) and a FAM-labelled oligonucleotide with the COUP-TFII-binding site from Snail-1. For supershift assay, anti-COUP-TFII antibody. (D) After ChIP with anti-Snail-1 using transfected LOVO cells as in C, target genes were detected by quantitative real-time PCR.