p73 is a potent inducer of IGFBP4 gene expression in hepatocellular carcinoma

Background: p53-family members p53, p63 and p73 are known for their involvement in regulating cell cycle, cell senescence and apoptosis. In their role as transcription factors and depending on their splice variants – with transactivation domain (TA) or dominant negative (DN) – p53 and its siblings activate or inhibit the transcription of specific target genes. We previously identified the gene for Insulin-like Growth Factor Protein 4 (IGFBP4) as potential p53-family target gene with prognostic relevance in hepatocellular carcinoma (HCC). In contrast to p53, the IGF system takes part in tissue growth and cell survival. IGFBP4 acts as inhibitor limiting IGF effects suggesting a possible interaction with p53 affairs. Considering this feature, we aimed to characterize the regulatory influence of p53 family members on IGFBP4.

Methods: Hep3B cells were transfected with rAd-p53, -TAp63, -TAp73, -DNp63, and -DNp73. Transcriptional regulation of IGFBP4 was determined by real time qPCR. Intra- and extracellular IGFBP4 protein levels were examined by Western Blotting and ELISA. TRANSFAC database analysis was performed to identify potential p53-family binding sites in the IGFBP4 locus. Identified sequences were cloned, deleted and analyzed in luciferase reporter assays to evaluate binding of p53-family members.

Results: IGFBP4 expression was increased by more than 30-fold in TAp73-transfected Hep3B cells, by more than 15-fold in DNp63- and by 3-fold in p53-transfected cells. Induction of intracellular IGFBP4 protein was detected in all transfected Hep3B cells, whereas extracellular IGFBP4 levels were only measurable after TAp73 and DNp63 transfection. Database analysis identified 2 putative p73 binding sites within intron 1 of the IGFBP4 gene. Intron 1-dependent luciferase activity was increased by up to 20-fold in TAp73-transfected cells. This induction was reduced by up to 70% when one of the putative binding sites was deleted.

Conclusion: These results identify the IGF inhibitor IGFBP4 as novel target gene for TAp73 in HCC. By demonstrating for the first time the interaction of TAp73 and IGFBP4 we enhance our knowledge in a so far unknown association of p53-family network and IGF signaling. Since in an independent study we identified IGFBP2 as an additional p53-family target gene, these results highlight the link between p53-family-mediated tumor-inhibiting mechanisms and IGF-dependent cell proliferation. We therefore suppose that the particular balance of these pathways decides on growth, cancerogenesis and treatment response.