IGFBP2 – a novel p53-family target gene in hepatocellular carcinoma

Background: In a variety of tumors, among these hepatocellular carcinoma (HCC), p53 family members exert cancerogenic or tumorsuppressive effects. p53 transcription factors (p53, p63, p73) react to cellular stress by transcriptionally regulating a specific set of genes. Target genes can be activated or inhibited depending on the particular p53 splice variants – with transactivation domain (TA) or dominant negative (DN) – and the characteristics of the specific binding site. We previously identified IGFBP2 (Insulin-Like Growth Factor Binding Protein 2) as a putative p53-protein target gene with prognostic relevance in HCC. The aim of this study was to elucidate how IGFBP2 is regulated by p53 family members.

Methods: Hep3B cells were transfected with rAd-p53 and -TAp73. Transcriptional regulation of IGFBP2 was determined by qPCR. Western Blot and ELISA were used to investigate intra- and extracellular protein levels of IGFBP2. Transfac database analysis was performed to identify potential p53-family binding sites in the IGFBP2 locus. These sequences were then cloned, mutated and evaluated by luciferase reporter assays to confirm p53 binding.

Results: TAp73-transfection induced a more than 25-fold increased IGFBP2 expression. A remarkable increase in intra- and extracellular IGFBP2 protein levels after TAp73-transfection was measurable as in controls no IGFBP2 was detectable. Transfection with TAp53 resulted in an up to 7-fold increased IGFBP2 expression. Two putative p53 and p73 binding sites (BS) are located within the promoter region. Another five putative BS for p73 and one for p53 were identified within intron 1 of the IGFBP2 locus. In TAp73-transfected cells intron1-dependent luciferase activity was increased by up to 150-fold, in TAp53 transfected cells by up to 20-fold. The identified p53 BS in intron 1 was confirmed, since after mutation and deletion of the latter luciferase activity was reduced by up to 90% in TAp53 and TAp73 transfected cells. Deletion of one of the putative p73 BS resulted in a reduction of luciferase activity by 85% after TAp73 transfection.

Conclusion: These results clearly identify IGFBP2 as a novel target gene for TAp73 and TAp53 in HCC. By demonstrating for the first time the interaction between TAp73 and IGFBP2 signaling we succeeded in finding an important, formerly unknown, link between p53-family network and the IGF axis. p53-family members exert tumor-inhibiting effects whereas the IGF axis is of crucial importance for cell proliferation. It is suggestive that the balance of these two signaling pathways influences tumor characteristics as well as treatment response. Therefore, apart from enhancing our understanding of cancerogenic processes in HCC our results indicate that fine-tuning of these pathways might offer new therapeutic options in HCC, an entity with so far limited therapeutic measures.