TY  - THES
A1  - Hauck, Stefanie
T1  - Mechanistic studies on FUSE binding protein 1 (FUBP1) inhibition in cancer cells
N2  - FUSE Binding Protein 1 (FUBP1) is a transcriptional regulator, which is overexpressed in various cancer entities, including hepatocellular carcinoma (HCC) and colorectal cancer (CRC). It fulfills pro-proliferative and anti-apoptotic functions in cancer cells, resulting in increased proliferation and reduced sensitivity towards apoptotic stimuli.
Previously, camptothecin (CPT) and its clinically used analog 7-ethyl 10hydroxycamptothecin (SN-38) were shown to inhibit FUBP1 in biophysical interaction displacement assays (AlphaScreen; surface plasmon resonance, SPR), and first insights into the cellular effects of FUBP1 inhibition were obtained. CPT and SN-38 are known to potently inhibit topoisomerase 1 (TOP 1), and until today, these inhibitors were thought to be specific for this target. This could be disproved by our FUBP1 binding studies. An open issue, which is addressed in this thesis, was the contribution of FUBP1 inhibition to SN-38-mediated apoptosis apoptosis.
During this thesis, a low micromolar efficacy of CPT/SN-38-induced inhibition of FUBP1 binding to the Far Upstream Sequence Element (FUSE) oligonucleotide of p21 was determined. Furthermore, FUBP1 was for the first time shown to directly interact with a potential FUSE sequence upstream of the transcription start in pro-apoptotic gene BIK. In proof of-principle experiments, an effective inhibition of the binding of FUBP1 to the FUSE BIK DNA by CPT/SN-38 was verified.
One of the main goals of this thesis was to further elucidate the contribution of cellular FUBP1-inhibition by CPT/SN-38 to the anti-cancer potential of these substances. For this purpose, the TOP 1 mutant and TOP 1 wild type colorectal cancer sub-cell lines HCT116 G7 and HCT116 S were used. CPT/SN-38 was shown to induce apoptosis in single and combinatorial treatments with mitomycin c (MMC), independently of the TOP 1 mutation status of the cells. Furthermore, a prominent induction of a FUBP1 target gene signature was observed upon treatment of both cell lines with CPT/SN-38. Consequently, CPT/SN-38 was able to fulfill its anticancer effects in these cells, although TOP 1 could not be the main target in the mutant cell line.
In a second approach to gain indirect evidence for FUBP1 dependent effects of CPT/SN-38, the TOP 1-specific inhibitors topotecan (TTN) and β lapachone (BL) were used for the treatment of HCC and CRC cell lines. Interestingly, the TOP 1 inhibitors TTN and BL exhibited a reduced potency in apoptosis induction compared to the dual (FUBP1 and TOP 1) inhibitor SN-38.
Finally, two independent screens for a specific FUBP1 inhibitor were performed. In the first approach, a small number of structural and functional CPT-derivatives that exhibited a reduced inhibitory potential against TOP 1, were tested for their ability to interfere with the FUBP1/FUSE binding. Two particular indenoisoquinoline derivatives revealed potent in vitro inhibition of FUBP1 with low micromolar IC50 values.
In a second approach, previously identified candidate FUBP1 inhibitors that had been isolated from the Maybridge Hit Finder library served as lead structures for a structure activity relationship (SAR) study of the inhibition of FUBP1 binding to the FUSE oligonucleotide. After two cycles of optimization, a medium-potent FUBP1 inhibitor was obtained that induced effective deregulation of FUBP1 target genes in cell culture experiments.
N2  - Der transkriptionelle Regulator "FUSE Binding Protein 1" (FUBP1) fungiert in verschiedenen Tumorentitäten, unter anderem dem hepatozellulären Karzinom (HCC) und dem kolorektalen Karzinom (CRC), als Proto-Onkogen. Es konnte gezeigt werden, dass die "short hairpin" (sh-RNA)-induzierte Herrunterregulation von FUBP1 in Krebszellen zu einer verringerten Proliferationsrate und einer erhöhten Sensitivität gegenüber apoptotischen Stimuli, beispielsweise Mitomycin C (MMC) oder Doxorubicin (DOX), führt...
Y1  - 2017
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/43120
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-431200
ER  -