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

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.