The role of E2F1 in cholangiocarcinoma - Potential therapeutic options

Cholangiocarcinoma is a very aggressive form of tumor, which still has a very poor prognosis, due in part to the often late time of diagnosis and the limited response to current forms of therapy. Therefore, it is necessary to work on new therapeutic approaches to improve the therapy and consequently the prognosis of CCA. This work was focused on the transcription factor E2F1, which is known to play a key role in cell cycle progression through its position in the Rb-pathway. As being a master regulator, E2F1 has a lot of target genes with complex interactions. By occupying such a key position in cell division and proliferation, E2F1 is also important in the proliferation of tumor cells. In many tumor entities an overexpression of E2F1 has already been described, although the role seems to be complex with contradictory results. In principle, this work can be divided into two experimental areas, based on the question of whether E2F1 can be used therapeutically in CCA: On the one hand, human CCA samples were examined, always in direct comparison with healthy liver tissue from identical patients. Here, the expression of E2F1 and several target genes (Cyclin E, Cyclin B1, MCM2, MCM7, c-Myc, Cyclin D1, SKP2 and FOXM1) was investigated. In conclusion, no meaningful results could be obtained at the protein level; there were no clear expression differences between tumor tissue and tumor-free tissue. At the RNA level, clearer differences could be obtained in some cases, whereby the expected correlation could only be confirmed for E2F1 itself and MCM2. Most of the other target genes even showed increased expression patterns in healthy tissue, concluding that the investigation of 33 CCA tumor samples did not lead to support the hypothesis that E2F1 is a promising therapeutic target. The second major experimental complex involved cell culture experiments with four CCA cell lines. In order to demonstrate the dependence of target genes on E2F1, E2F1 was suppressed by silencing and then the expression of target genes underneath was examined. From these results, it could be deduced that suppression of E2F1 significantly affects the expression of the target genes. Suppression over 50% starting from the expression in the control groups was detected here for MCM2,MCM7, SKP2 and RRM2. Furthermore, the cell lines were treated with the E2F1 inhibitor HLM006474 at different concentration levels and subsequently their growth behavior was monitored. Basically, based on this experimental model, it can be concluded that the application of HLM006474 has effects on the growth behavior of the CCA cell lines, and the difference between 50 and 100 µM was mostly not large anymore. Together with the results from the silencing experiments, it can be supposed that a therapeutic inhibition of E2F1 is possible and should also affect some genes involved in tumorigenesis and proliferation. Finally, the results of this work can be summarized by the fact that the investigation of the human CCA samples, although not allowing clear conclusions about the role of E2F1 and its target genes in CCA, the cell culture experiments nevertheless showed that E2F1 in CCA has an influence on the expression of the other target genes. Inhibition of E2F1 by HLM006474 affects growth behavior, so the results provide at least a starting point that the use of HLM006474 should be further investigated and new experimental models should be tested in vitro and in vivo and also should be expanded to include combination with already established CCA therapies to see if clinical use of HLM006474 could achieve good results in tumor treatment and if its use in humans would eventually be feasible