Upregulation of TCF21 inhibits migration of adrenocortical carcinoma cells

Abstract

Background: Adrenocortical carcinomas (ACC) are rare and aggressive cancer. Our previous study has revealed that the transcription factor 21, TCF21, is downregulated in ACC and regulates steroidogenic factor 1 (SF-1) binding to the SF-1 E-box promoter. In addition, it could be found that TCF21 is a predictor of overall survival (OS) in adult carcinomas.

Methods: In this study, it was investigated the correlation between TCF21 expression and the promoter methylation status in adrenocortical tumor cells, carcinomas and adenoma. The biological function and potential molecular mechanism of TCF21 restoration in migration and invasion of ACC cells was examined.

Results: We could be demonstrated a negative correlation between the level of TCF21 expression and methylation of its promoter in adenoma and carcinoma cells indicating the epigenetic control of TCF21 expression. It was also demonstrated that the expression of TCF21 inhibits migration and invasion in the ACC cell line, H295R cells, using plasmid transfection to express TCF21. Furthermore, it could be investigated the TCF21 function as tumor suppressor probably through Kisspeptin 1 (KISS-1) expression and epithelial-mesenchymal transition (EMT) reversion, as well as the modulation of several metalloproteinases in ACC cells.

Conclusions: Our results suggest that enhancement of TCF21 expression levels may be a potential strategy to revert invasive abilities in adrenocortical carcinomas.

Keywords: Adrenocortical carcinoma cells; Matrix metalloproteinases; Methylation; Migration; TCF21.

Fig. 1

TCF21 expression and promotor methylation. A Relative TCF21 expression in adrenocortical tumor cells; B Methylation levels of TCF21 promoter; C Negative Pearson’s correlation between TCF21 expression and level of promoter methylation (r = − 0.9984). NA normal adrenal pool; ACC-T36 adrenocortical carcinoma cell culture from patient; ACAPed-T7, pediatric adrenocortical adenoma cell culture from patient; H295R cell line. The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by One-way ANOVA

Fig. 2

Modulation of TCF21 expression. A TCF21 overexpression in H295R transfected with pCMVMycPod1 or empty vector (pCMVMyc); B Inhibition of TCF21 in ACAPed-T7 cells using siRNATCF21 or control (siRNA). The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 3

Upregulation of TCF21 decrease ACC cell migration and invasion. A Migration of H295RpCMVMycPod1 cells (TCF21) was inhibited in relation to respective controls and representative stained membranes; B Invasion of H295pCMVMycPod1 cells (TCF21) was inhibited in relation to respective controls and representative stained membranes. The experiments were performed in triplicate and repeated three times. Magnification of membranes (100×). The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 4

Downregulation of TCF21 promote ACA migration and invasion. A, C Migration of ACAPed-T7siRNATCF21 cells in relation to control (ACAPed-T7siRNA); B, D Invasion of ACAPed-T7siRNATCF21 cells in relation to control (ACAPed-T7siRNA). The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 5

TCF21 increased the expression of anti-invasive effectors in ACC cells. A Relative mRNA level and protein expression of metalloproteinase 8 (MMP-8) and representative immunoblot; B Relative mRNA level of metalloproteinase inhibitor 1 (TIMP-1); C Relative mRNA level of tumor metastasis suppressor 1 KISS-1, in H295R/TCF21 cells and respective controls. The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 6

TCF21 downregulated the invasive effectors in ACC cells. A Relative mRNA level and protein expression of metalloproteinase 9 (MMP-9) and representative immunoblot; B Relative mRNA level of metalloproteinase 14 (MMP-14); C Relative mRNA level of metalloproteinase 2 (MMP-2), D Relative mRNA level and protein expression of Vimentin (VIM) and representative immunoblot in H295R/TCF21 cells and respective controls. The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 7

Inhibition of TCF21 inhibited MMP-8 and promote MMP-9 expression. A Relative mRNA level and protein expression of metalloproteinase 8 (MMP-8) and with representative immunoblot experiment; B Relative mRNA level and protein expression of metalloproteinase 9 (MMP-9) and representative immunoblot in ACAPed-T7siRNATCF21. The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 8

Overexpression of metalloproteinase MMP-8 in adrenocortical carcinoma cells. A MMP-8 expression in H295R cells transfected with pCMVMMP-8 and empty vector; representative immunoblot of MMP-8 expression; B migration and invasion assays in H295R transfected with pCMVMMP-8 and representative stained membranes. The experiments were performed in triplicate and repeated three times. Magnification of membranes (100×). The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 9

Overexpression of metalloproteinase MMP-9 in adrenocortical carcinoma cells. A MMP-9 expression in H295R cells transfected with pcDNAMMP-9 and empty vector; representative immunoblot of MMP-9 expression; B migration and invasion assays in H295R transfected with pcDNAMMP-9 and representative stained membranes. The experiments were performed in triplicate and repeated three times. Magnification of membranes (100×). The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

Fig. 10

Overexpression of metalloproteinase in adrenocortical adenoma cells. A MMP-9 expression in ACAPed-T7 transfected with pcDNAMMP-9 and empty vector; representative immunoblot experiment of MMP-9; B migration and invasion assays in ACAPedT7 transfected with pcDNAMMP-9 and representative stained membranes. Magnification of membranes (100×). The experiments were performed in triplicate and repeated three times. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test