The homeobox transcription factor Prox1 inhibits proliferation of hepatocellular carcinoma cells by inducing p53-dependent senescence-like phenotype

Abstract

The homeobox transcription factor Prox1 is highly expressed in adult hepatocytes and is involved in the regulation of bile acid synthesis and gluconeogenesis in the liver by interacting with other transcriptional activators or repressors. Recent studies showed that Prox1 could inhibit proliferation of hepatocellular carcinoma (HCC) cells and reduced Prox1 expression was associated with poor prognosis of HCC patients. However, the underlying mechanism by which Prox1 attenuates HCC growth is still unclear. In this study, we demonstrated that Prox1 induced senescence-like phenotype of HCC cells to reduce cell proliferation. Our results indicated that the tumor suppressor p53 is a key mediator of Prox1-induced growth suppression because Prox1 only induced senescence-like phenotype in HCC cells harboring wild type p53. In addition, knockdown of p53 by shRNA reversed the effect of Prox1. However, chromatin immunoprecipitation assay did not demonstrate the direct binding of Prox1 to proximal promoter of human p53 gene suggesting Prox1 might not directly activate p53 transcription. We found that Prox1 suppressed Twist expression in HCC cells and subsequently relieved its inhibition on p53 gene transcription. The involvement of Twist in the regulation of p53 by Prox1 was supported by the following evidence: (1) Prox1 inhibited Twist expression and promoter activity; (2) knockdown of Twist in SK-HEP-1 cells upregulated p53 expression and (3) ectopic expression of Twist counteracted Prox1-induced p53 transcription and senescence-like phenotype. We also indentified an E-box located at p53 promoter which is required for Twist to inhibit p53 expression. Finally, our animal experiment confirmed that Prox1 suppressed HCC growth in vivo. Collectively, we conclude that Prox1 suppresses proliferation of HCC cells via inhibiting Twist to trigger p53-dependent senescence-like phenotype.

Keywords: Prox1; Twist; hepatocellular carcinoma; p53; senescence.

Figure 1. Prox1 induces cell senescence-like phenotype via p53 induction. (A) SK-HEP-1 cells were transfected with pcDNA (C) or Prox1 (P) expression vector. After different times, DNA synthesis was assessed by using BrdU incorporation assay and the number of cells with positive staining of the control group was defined as 100%. *p < 0.05 (B) Senescence-like phenotype was assessed by β-galactosidase staining at 72 h after transfection and typical microscope figures were shown. (C) Expression of various CDKIs in control and Prox1-overexpressing cells was studied by western blotting. (D) SK-HEP-1 cells were co-transfected with pcDNA (Con) or Prox1 with luciferase (Luc) or p53 shRNA for 48 h. β-galactosidase-positive cell number of the group transfected with pcDNA and luciferase shRNA was defined as 1. *p < 0.05. Protein level of p53 was studied by western blotting. (E) SK-HEP-1 cells were co-transfected with pcDNA (Con), Prox1 expression vector, luciferase (Luc) or p53 shRNA for 48 h. Expression of hTERT and CXCL1 was examined by RT-PCR.

Figure 2. Prox1 did not increase β-galactosidase staining in HCC cells with p53 deficiency. p53-deleted Hep3B cells or p53-mutated Mahlavu cells were transfected with pcDNA (Con) or Prox1 expression vector. After 72 h, cells were subjected to β-galactosidase staining and Prox1 protein level was studied by western blotting.

Figure 3. Inhibition of Twist by Prox1 is required for the upregulation of p53. (A) SK-HEP-1 cells were trasnfected with pcDNA or Prox1 expression vector. After 48 h, ChIP assay was performed by using anti-Prox1 or anti-Twist antibody to detect the binding of these two proteins to human p53 proximal promoter region. (B) Effect of Prox1 on Twist expression was investigated by western blotting. (C) SK-HEP-1 cells were co-transfected human p53 promoter with luciferase (Luc) or Twist shRNA for 48 h. Protein levels of Twist and p53 (upper panel) and luciferase activity (bottom panel) were determined. Promoter activity of cells co-transfected with luciferase shRNA was defined as 100%. *p < 0.05 when compared with the control group. (D) SK-HEP-1 cells were transfected with pcDNA (Con), Prox1 or Prox+Myc-tag Twist vector. After 48 h, RT-PCR and western blotting were performed to study Prox1, Twist and p53 expression.

Figure 4. Twist inhibits p53 expression via E-box in the promoter. (A) HepG2 cells were co-transfected p53 promoter with pcDNA (Con) or Myc-tag Twist vector. After 24 h, p53 promoter activity and protein level were determined. (B) HepG2 cells were transfected with luciferase (as a control) or Prox1 shRNA. Protein and mRNA levels of Prox1, Twist and p53 were studied. (C) HepG2 cells were co-transfected human wild type (WT) or E-box mutant of human p53 promoter with pcDNA (Con) or Myc-tagged Twist for 48 h and luciferase activity was determined. Promoter activity of cells co-transfected with pcDNA was defined as 100%. *p < 0.05 when compared with the control group. (D) SK-HEP-1 cells were transfected with wild-type or E-box mutant p53 promoter and promoter activity was determined.

Figure 5. Prox1-induced senescence-like phenotype and inhibition of anchorage- independent growth were abolished by Twist. (A) SK-HEP-1 cells were transfected with pcDNA (Con), Prox1 or Myc-tagged Twist expression vectors. β-galactosidase staining was performed and the number of positive signal of the control group was defined as 1. *p < 0.05. (B) SK-HEP-1 cells were trasnfected with control, Prox1 or Twist expression vectors. Colonies were counted and the number of the control group was defined as 100%.*p < 0.05. (C) HepG2 cells were trasnfected with luciferase (Luc) or Prox1 shRNA. Colonies were counted and the number of control group was defined as 1. *p < 0.05.

Figure 6. Prox1 inhibits tumor growth in vivo. (A) SK-HEP-1 cells were transfected with pcDNA or Prox1 expression vector and subjected to subcutaneous injection as described in Materials and Methods. Tumor growth was continuously monitored and mice were sacrificed at 14 d after injection. Tumors were removed and weighted. *p < 0.05 when compared with the control group. (B) Immunohistochemical staining of tumor tissues (upper panel) and immunocytochemical staining of control and Prox1- expressing SK-HEP-1 cells (bottom panel) were conducted. Representative images were shown. (C) Tumor tissue sections were stained with K_i-67, Twist and p53 antibodies and representative images were shown.