PRRX1 deficiency induces mesenchymal-epithelial transition through PITX2/miR-200-dependent SLUG/CTNNB1 regulation in hepatocellular carcinoma

Abstract

Metastasis is a major obstacle to better prognosis in patients with hepatocellular carcinoma (HCC). Mesenchymal-epithelial transition (MET) is the driving force for metastatic colonization in which E-cadherin re-expression is a critical procedure. It has been reported that the loss of paired-related homeobox transcription factor 1 (PRRX1) is required for cancer cell metastasis. However, the role of PRRX1 in MET and how its downregulation triggers E-cadherin re-expression are unknown. In this study, we performed a systematic, mechanistic study regarding the role of PRRX1 in MET of HCC. We observed PRRX1 downregulation in HCC tissues, which correlated with early metastasis and short overall survival. Overexpression of PRRX1 induced epithelial-mesenchymal transition (EMT), but did not promote metastasis formation, while knockdown of PRRX1 promoted metastasis and colonization of circulating HCC cells as shown in animal model. PRRX1 protein levels reversely correlated with E-cadherin levels in HCC cell lines. PRRX1 knockdown promoted E-cadherin re-expression and cell proliferation and inhibited cell invasion and migration. The microarray results showed that PRRX1 deficiency regulated extracellular matrix (ECM) interaction, focal adhesion, TGF-β signaling and cancer pathways. PRRX1 knockdown upregulated paired-like homeodomain 2 (PITX2) and inhibited catenin beta 1 (CTNNB1) and SNAIL family zinc finger 2 (SLUG). Silencing of PITX2 reversed CTNNB1 and SLUG inhibition and E-cadherin re-expression. PITX2 upregulation increased miR-200a and miR-200b/429, which further inhibited the transcription of CTNNB1 and SLUG, respectively, thus abrogating the inhibitory effect on E-cadherin. In conclusion, our data showed that the downregulation of PRRX1 induced E-cadherin re-expression through PITX2/miR-200a/CTNNB1 and PITX2/miR-200b/429/SLUG pathway.

Keywords: E-cadherin re-expression; PITX2; PRRX1; hepatocellular carcinoma; mesenchymal-epithelial transition.

FIGURE 1

Paired‐related homeobox transcription factor 1 (PRRX1) was down‐regulated in hepatocellular carcinoma (HCC) and correlated with poor prognosis. A, Western blotting (WB) results of Prrx1 in 10 pairs of normal (N) liver tissue and peritumoral (P) and tumoral (T) tissues. B, Quantification and comparison of immunohistochemistry (IHC) staining scores of PRRX1 in 221 pairs of HCC tumor and peritumor tissues. C, Representative images of IHC staining of Prrx1 in peritumoral and tumoral tissues. D and E, Kaplan‐Meier plot of the disease‐free survival (DFS) and overall survival (OS) of patients with HCC, stratified by PRRX1 levels

FIGURE 2

Downregulation of paired‐related homeobox transcription factor 1 (PRRX1) promoted metastasis and colonization of circulating tumor cells. A, The interfering efficiency was verified by qPCR after Prrx1 shRNA transfection in HCCLM3 cells. **P < .01. B, The interfering efficiency was verified by Western blotting (WB) after Prrx1 shRNA transfection in HCCLM3 cells. C, Comparison of the tumor volumes in the livers of nude mice injected with HCCLM3 cells transfected with control shRNA or PRRX1 shRNA using an in situ tumor model (n = 12 in each group). *P < .05. D, Representative images of metastatic lung nodules in the in situ liver tumor model. *P < .05. E. Representative images of lung tissue sections from the in situ liver tumor model. F. Two mice injected with HCCLM3 cells with PRRX1 knockdown developed subcutaneous metastasis in the in situ liver tumor model. G. Comparisons of overall survival (OS) curves in a lung metastatic colonization model of mice injected with HCCLM3 cells transfected with control shRNA or PRRX1 shRNA (n = 14 in each group). H, Comparison of pulmonary metastasis in the lung metastatic colonization model. *P < .05. I, Representative images of lung tissue sections from the metastatic colonization model

FIGURE 3

Downregulation of paired‐related homeobox transcription factor 1 (PRRX1) induced mesenchymal‐epithelial transition (MET) in hepatocellular carcinoma (HCC) cells. A, Western blotting (WB) results of E‐cadherin and PRRX1 in HCC cell lines. B, Correlation between the relative density of E‐cadherin and PRRX1 bands in HCC cell lines (Pearson r = −0.8949, P = .0403). C, WB results of E‐cadherin in HCC cells with Prrx1α or Prrx1β overexpression. D, Immunofluorescent staining of E‐cadherin in HepG2 cells with Prrx1α or Prrx1β overexpression. E, WB results of E‐cadherin in HCC cells with PRRX1 knockdown. F. Immunofluorescent staining of E‐cadherin in HCCLM3 cells with PRRX1 knockdown. G, WB results of β‐catenin in HCC cells with PRRX1 knockdown. H, Cell invasion was tested using transwell Matrigel invasion assays after PRRX1 knockdown in HCC cells. The quantitation of cell invasion is in the right panel. *P < .05. I, Cell migration was tested using wound healing assays after PRRX1 knockdown in HCC cells. The quantitation of cell migration is in the right panel. *P < .05. J, Cell proliferation was tested using a colony formation assay after PRRX1 knockdown. *P < .05, **P < .01. K, WB results of cyclin D1 and cyclin‐dependent kinases (CDKs) in HCC cells with PRRX1 knockdown

FIGURE 4

Downregulation of paired‐related homeobox transcription factor 1 (PRRX1) enriched mesenchymal‐epithelial transition (MET)‐related pathways. A, Heat map of the most differentially expressed genes between three pairs of control cells and HCCLM3 cells with PRRX1 knockdown. B, Ten most changed pathways between control cells and HCCLM3 cells with PRRX1 knockdown. C, The gene network plot based on the top four pathways. D and E, qPCR results for SNAIL family zinc finger 2 (SLUG), catenin beta 1 (CTNNB1), transforming growth factor beta receptor 2 (TGFBR2), SMAD family member 6 (SMAD6), SMAD family member 1 (SMAD1), E‐cadherin 1 (CDH1), collagen type III alpha 1 chain (COL3A1), and paired‐like homeodomain transcription factor 2 (PITX2) in HCC cells with PRRX1 knockdown. *P < .05

FIGURE 5

Inhibition of paired‐like homeodomain transcription factor 2 (PITX2) reverses paired‐related homeobox transcription factor 1 (PRRX1) deficiency–induced decreases in SNAIL family zinc finger 2 (SLUG) and catenin beta 1 (CTNNB1) and increase in E‐cadherin. A, Western blotting (WB) results of PITX2 in control cells and hepatocellular carcinoma (HCC) cells with PRRX1 knockdown. B, WB results of PITX2 in control HCC cells and HCC cells overexpressing PRRX1. C, GFP was immunoprecipitated with a GFP antibody and then analyzed by WB using a PITX2 antibody. D, PITX2 was immunoprecipitated with a PITX2 antibody and then analyzed by WB using a PRRX1 antibody. E and F, PITX2 in HCC cells transfected with control or PRRX1 shRNA was silenced by siRNA; then, PITX2, E‐cadherin, SLUG, and CTNNB1 mRNA levels were detected by qPCR. *P < .05, **P < .01. G, PITX2 in HCC cells transfected with control or PRRX1 shRNA was silenced by siRNA; then, E‐cadherin, SLUG, and β‐catenin protein levels were detected by WB

FIGURE 6

Paired‐like homeodomain transcription factor 2 (PITX2) upregulates miR‐200s, which suppress catenin beta 1 (CTNNB1) and SNAIL family zinc finger 2 (SLUG) expression. A, qPCR analyses of miR‐200 family members in hepatocellular carcinoma (HCC) cells with paired‐related homeobox transcription factor 1 (PRRX1) knockdown. *P < .05. B, PITX2 in HCC cells transfected with control or PRRX1 shRNA was silenced by siRNA; then, miR‐200 family members were detected by qPCR. *P < .05. C, miR‐200a target sites in the 3′‐UTR of CTNNB1 and miR‐200b/miR‐429 target sites in the 3′‐UTR of SLUG. D and E, HCCL cells with PRRX1 knockdown were transfected with a miR‐200a inhibitor; then, CTNNB1 was detected by qPCR and Western blotting (WB). *P < .05. F and G, HCC cells with PRRX1 knockdown were transfected with the miR‐200a mimic, and CTNNB1 was detected by qPCR and WB. H and I, HCC cells with PRRX1 knockdown were transfected with a miR‐200b or miR‐429 inhibitor, and SLUG was detected by qPCR and WB. J and K, HCC cells with PRRX1 knockdown were transfected with a miR‐200b or miR‐429 mimic, and SLUG was detected by qPCR and WB. L and M, Normalized luciferase activity of the 3′‐UTR SLUG gene luciferase reporter (wild‐type SLUG) with control miRNA or pre‐miR‐200a (L)/pre‐miR‐429 (M) shows a loss of luciferase activity upon the expression of miR‐200a and miR‐429. *P < .05. N, Diagram showing PRRX1 deficiency–induced mesenchymal‐epithelial transition (MET) in HCC