Abstract
The molecular causes by which the epidermal growth factor receptor tyrosine kinase induces malignant transformation are largely unknown. To better understand EGFs' transforming capacity whole genome scans were applied to a transgenic mouse model of liver cancer and subjected to advanced methods of computational analysis to construct de novo gene regulatory networks based on a combination of sequence analysis and entrained graph-topological algorithms. Here we identified transcription factors, processes, key nodes and molecules to connect as yet unknown interacting partners at the level of protein-DNA interaction. Many of those could be confirmed by electromobility band shift assay at recognition sites of gene specific promoters and by western blotting of nuclear proteins. A novel cellular regulatory circuitry could therefore be proposed that connects cell cycle regulated genes with components of the EGF signaling pathway. Promoter analysis of differentially expressed genes suggested the majority of regulated transcription factors to display specificity to either the pre-tumor or the tumor state. Subsequent search for signal transduction key nodes upstream of the identified transcription factors and their targets suggested the insulin-like growth factor pathway to render the tumor cells independent of EGF receptor activity. Notably, expression of IGF2 in addition to many components of this pathway was highly upregulated in tumors. Together, we propose a switch in autocrine signaling to foster tumor growth that was initially triggered by EGF and demonstrate the knowledge gain form promoter analysis combined with upstream key node identification.
Highlights
Epidermal growth factor is an important mitogen for hepatocytes for its ability to modulate proto-oncogene as well as liver specific gene expression
Transcription of a gene is determined to a major part by the activity of transcription factors, which in turn recognize specific short DNA segments, i.e. transcription factor binding sites (TFBSs) which are often situated in the promoter region upstream of the transcription start site (TSS)
Analysis of TF co-occurrences in promoters of upregulated tumor genes To further investigate the EGF-induced switch from transgenic to tumor state, we performed another step of promoter analysis, where we focused on TFs, whose regulation of activity can be mechanistically explained by signal transduction cascades downstream of an EGF receptor ligand or an EGF receptor
Summary
Epidermal growth factor is an important mitogen for hepatocytes for its ability to modulate proto-oncogene as well as liver specific gene expression. The here described and newly developed method focuses on the identification of transcription factor binding sites with co-occupancy in the promoters of differentially expressed genes in a statistically significant manner This enabled hypotheses generation and an identification of transcription factors acting on such a promoter set with the ultimate goal to identify ‘‘molecular triggers’’ in gene regulatory networks forcing hepatocytes into malignant transformation. In an effort to search for key molecules in the signalling network upstream of the identified transcription factors the insulin-like growth factor pathway was identified that may represent a molecular switch from the EGF receptor tyrosine kinase route to the tumour state thereby rendering malignantly transformed cells independent of EGF receptor activity Further evidence for this hypothesis was obtained when the gene expression of IGF2 and its down stream partners was investigated and determined to be highly significantly induced in tumour cells as were many components of this pathway
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