TCF7L2 promotes anoikis resistance and metastasis of gastric cancer by transcriptionally activating PLAUR

Abstract

Gastric cancer (GC) is the most common gastrointestinal malignant tumor, and distant metastasis is a critical factor in the prognosis of patients with GC. Understanding the mechanism of GC metastasis will help improve patient prognosis. Studies have confirmed that urokinase-type plasminogen activator receptor (PLAUR) promotes GC metastasis; however, its relationship with anoikis resistance and associated mechanisms remains unclear. In this study, we demonstrated that PLAUR promotes the anoikis resistance and metastasis of GC cells and identified transcription Factor 7 Like 2 (TCF7L2) as an important transcriptional regulator of PLAUR. We also revealed that TCF7L2 is highly expressed in GC and promotes the anoikis resistance and metastasis of GC cells. Moreover, we found that TCF7L2 transcription activates PLAUR. Finally, we confirmed that TCF7L2 is an independent risk factor for poor prognosis of patients with GC. Our results show that TCF7L2 and PLAUR are candidate targets for developing therapeutic strategies for GC metastasis.

Keywords: anoikis; gastric cancer; transcription; transcription Factor 7 Like 2; urokinase-type plasminogen activator receptor.

Figure 1

Relationship between anoikis resistance and metastasis of cancer cells.

Figure 2

PLAUR promotes the malignant phenotype of GC cells. a-b Effects of PLAUR knockdown or overexpression on the proliferation of GC cells, measured by CCK-8 (a) and EdU assay (b). c After GC cells were suspended for 24h, the effects of PLAUR knockdown or overexpression on the anoikis resistance of GC cells, analyzed via flow cytometry. d Changes in apoptosis-related proteins after PLAUR knockdown or overexpression were analyzed via western blot in GC cells suspended for 24 h. e-f Effects of PLAUR knockdown or overexpression on the migration of GC cells, evaluated by transwell (e) and wound healing assay (f). Compared with the corresponding control group, * P < 0.05, ** P < 0.001.

Figure 3

PLAUR promotes the GC process in vivo. a Knockdown of PLAUR inhibited the growth of subcutaneous xenograft tumors in nude mice. The bioluminescence images of the subcutaneous tumors were displayed in a small animal in vivo imaging system, and the tumor growth curve for 24 days was measured. b Gross specimen and average weight of the tumor after sacrifice. Knockdown of PLAUR inhibited the volume and weight of subcutaneous xenograft tumors in nude mice. c Histological characteristics and Ki67 expression of the tumor were examined via H&E staining and IHC, respectively. The knockdown of PLAUR inhibited the Ki67 expression of subcutaneous xenograft tumors in nude mice. d Knockdown of PLAUR inhibited lung metastasis in nude mice. The bioluminescence image of lung metastases and the count of lung metastatic nodules are displayed, 28 days after tail veins were injected with GC cells. The white arrows represent the lung metastasis nodules. e Lung metastasis tumors were confirmed via H&E staining. Compared with the control group, * P<0.001.

Figure 4

TCF7L2 is a potential transcriptional regulator of PLAUR. a Silver staining of PLAUR binding protein identified in DNA pulldown. b Heat map of expression of transcription factors of PLAUR in GC samples and normal samples. c TCF7L2, FOXP3, and E2F1 were identified as transcription factors of PLAUR through LC-MS/MS, combined with the screening results of the PROMO database. d Expression correlations of TCF7L2, FOXP3, E2F1, and PLAUR were analyzed based on TCGA-seq data. TCF7L2 has the highest correlation with PLAUR expression. e TCF7L2 protein was confirmed to be present in the pull-down product by western blot.

Figure 5

TCF7L2 promotes transcription of PLAUR in GC. a Representative image of the co-localization of TCF7L2 protein (green) and PLAUR (red) in MKN45 cell lines observed by confocal microscopy. b Representative image of the co-localization of TCF7L2 protein (green) and PLAUR (red) in 15 GC tissues (cohort 1) observed via confocal microscopy; the expression of TCF7L2 and PLAUR was positively correlated via fluorescence quantitative analysis. c-d TCF7L2 has a positive regulatory effect on the expression of PLAUR in GC cells. The effects of TCF7L2 silencing on PLAUR mRNA or protein expression in GC cells as detected by RT-qPCR (c) and western blot (d), respectively. e Motif of the TCF7L2 binding site in the PLAUR promoter, predicted through the JASPAR dataset. f Schematic of the TCF7L2 binding site on the PLAUR promoter. g ChIP-qPCR analysis of TCF7L2 binding to the PLAUR promoter in MKN45 and 293T cell lines. The second position was the most important binding site. e Luciferase activity was determined after mutation of the second TCF7L2 site in the PLAUR promoter in MKN45 and 293T cell lines. The luciferase activity of the wild-type PLAUR promoter was significantly higher than that of mutant PLAUR promoter. Compared with the corresponding control group, * P < 0.001.

Figure 6

TCF7L2 promotes the malignant phenotype of GC cells. a-b Effects of TCF7L2 knockdown or overexpression on the proliferation of GC cells measured by CCK-8 (a) and EdU assay (b). c After GC cells were suspended for 24h, the effects of TCF7L2 knockdown or overexpression on the anoikis resistance of GC cells were analyzed via flow cytometry. d Changes in apoptosis-related proteins after TCF7L2 knockdown or overexpression were analyzed via western blot in GC cells suspended for 24 h. e-f Effects of TCF7L2 knockdown or overexpression on the migration of GC cells as evaluated by transwell (e) and wound healing assay (f). Compared with the corresponding control group, * P < 0.05, ** P < 0.001.

Figure 7

TCF7L2 promotes the GC process in vivo. a Knockdown of TCF7L2 inhibited the growth of subcutaneous xenograft tumors in nude mice. Bioluminescence images of the nude mice subcutaneous tumors were displayed through a small animal in vivo imaging system, and the tumor growth curve was measured for 24 days. b Gross specimen and average weight of the tumor after animals were sacrificed. Knockdown of TCF7L2 inhibited the volume and weight of subcutaneous xenograft tumors in nude mice. c Histological characteristics and Ki67 expression of the tumor were examined via H&E staining and IHC, respectively. Knockdown of TCF7L2 inhibited the Ki67 expression in subcutaneous xenograft tumors. d Knockdown of TCF7L2 inhibited lung metastasis. The bioluminescence image of lung metastases and the count of lung metastatic nodules was obtained 28 days after tail veins were injected with GC cells. The white arrows represent the lung metastasis nodule. e Lung metastases were confirmed via H&E staining (left); knockdown of TCF7L2 inhibited PLAUR expression in lung metastases evaluated by IHC (right). Compared with the control group, * P < 0.001.

Figure 8

PLAUR is essential for the malignant phenotype of GC cells mediated by TCF7L2. a-b Cell proliferation of MKN45 cells with knockdown of TCF7L2 or control, and re-expression of PLAUR in TCF7L2 depleted cells, as quantified by CCK-8 (a) and EdU (b) assay, respectively. c After GC cells were suspended for 24 h, the anoikis resistance of MKN45 cells with knockdown of TCF7L2 or control and re-expression of PLAUR in TCF7L2 depleted cells were assessed via flow cytometry. d After GC cells were suspended for 24 h, western blot for apoptosis-related proteins in MKN45 cells with the depletion of TCF7L2 or control, or re-expression of PLAUR in TCF7L2 depleted cells. e-f Measurement of cell migration by transwell (e) and wound healing (f) assays using MKN45 cells with depletion of TCF7L2 or control, or re-expression of PLAUR in TCF7L2 depleted cells. * P<0.05, ** P<0.001.

Figure 9

High TCF7L2 expression predicts poor prognosis of patients with GC. a Typical images of TCF7L2 protein in GC and adjacent tissues stained by IHC. In GC tissues, TCF7L2 protein was located in the nucleus and cytoplasm; in adjacent tissues, TCF7L2 protein was primarily located in cytoplasm. b Survival curve of 121 GC patients (cohort 2). High TCF7L2 expression correlated with poor prognosis of GC patients. c TCF7L2 protein was quantitatively analyzed via western blot in 10 pairs of fresh GC and adjacent tissues (cohort 3). d Independent prognostic factor for overall survival of 121 patients with GC, shown as a forest plot.

Figure 10

Schematic summary. Mechanistically, TCF7L2 is upregulated in GC and transcriptionally activates PLAUR, which promotes anoikis resistance and metastasis of GC cells.