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Abstract

Gallbladder cancer (GBC) and cholangiocarcinoma (CCA) comprise the group of biliary tract cancers (BTC). They are a rather rarely occurring group of gastrointestinal tumors, however, with bad prognosis. Due to the lack of specific symptoms and late diagnosis, the only potentially curative treatment is surgical resection, but only few patients are eligible, while the only treatment option for unresectable patients lies in general chemotherapy with a platinum-derived agent and gemcitabine. However, this is usually not extending the life expectancy substantially. BTCs are understudied cancers, of which the pathological mechanisms behind tumor formation are mostly unknown, but it is thought that chronic inflammatory processes can contribute to carcinogenesis. In preceding studies to the here presented doctoral thesis, miRNA profiling of a large German cohort of 40 GBC and 8 healthy gallbladder samples was performed. This led to the identification of 24 human miRNAs, which are differentially expressed between healthy gallbladder epithelium and gallbladder cancer. These miRNAs could further be classified into pro-survival and anti survival associated miRNAs, with miR 145 5p being the most downregulated miRNA and miR 4502 representing a potentially oncogenic miRNA. Furthermore, it was shown that CCA cells overexpressing miR 145 5p, significantly upregulated the STAT1 signaling pathway. This led to a tumor suppressive phenotype, as assessed by decreased colony formation and cell viability in CCA cells. Within the scope of this study, two projects were pursued: firstly, confirming the upregulation of STAT1 signaling by miR 145 5p and sustained tumor suppressive STAT1 signaling by concomitant downregulation of the phosphatase PTPRF and secondly: proteomic profiling of FFPE (formalin fixed paraffin embedded) GBC and healthy gallbladder samples of the same cohort as mentioned above, were analyzed by in-depth quantitative proteomics. This resulted in the identification of differentially expressed proteins, leading to the identification of the tumor suppressor FHL1, which is also a target of the oncogenic miRNA miR 4502. Regarding the first part, STAT1 gene and protein expression, as well as STAT1-induced target genes were investigated in CCA, GBC and hepatocellular carcinoma (HCC) cells under the ectopic overexpression of miR 145 5p. This showed that miR 145 5p induced STAT1 upregulation is functional and predominantly occurs in CCA. This emphasizes the molecular distinction between these cancer entities. Additionally, it was shown that along the axis of miR 145 5p and STAT1 activation, the phosphatase PTPRF is downregulated. PTPRF has been predicted to be a miR 145 5p target, however, proving the direct binding and subsequent downregulation was not successful so far. Nevertheless, as STAT1 and PTPRF co-precipitate, it is likely for PTPRF to be able to dephosphorylate STAT1, thereby negatively regulating STAT1 signaling. This led to the conclusion that the tumor suppressive miR 145 5p can lead to the induction of STAT1 expression, along with sustained phosphorylation by concomitant downregulation of PTPRF. Thereby, STAT1 signaling was further enhanced, since the attenuation of tumor suppressive signaling by PTPRF was decreased. These findings suggest the use of the miR 145 5p/STAT1/PTPRF axis to develop a targeted therapy for CCA. For the second part of the studies, a new proteomic approach from FFPE-tissues of GBC and healthy gallbladder was established. Up to date, only few studies using this technology exist as it still challenging. However, the successful performance of quantitative proteomic profiling with a deep resolution of differentially expressed proteins of the GBC cohort is described here. This created the possibility to integrate the miRNA array data with the protein expression data. It was found that FHL1 is one of the topmost downregulated proteins in GBC. Furthermore, miR 4502 is an upregulated miRNA in the short-survival group and it was predicted by miRNA-binding algorithms to target FHL1. These predictions were confirmed by a luciferase assay, proving direct binding of miR 4502 to the 3’ untranslated region (3’UTR) of FHL1. In addition, miR 4502 is downregulating FHL1 in GBC cells after overexpression of mimic 4502 and miR 4502 inhibitor is upregulating FHL1 levels. FHL1 exhibited tumor suppressive properties by reducing colony formation, cell proliferation and cell viability in GBC cells. FHL1 contains protein-protein interaction domains and it has previously been shown to associate with transcription factors, such as the cofactor RBPJ, without being capable of binding DNA itself. In this study, the binding of FHL1 to RBPJ was confirmed and thus FHL1 is likely to replace the intracellular domain of NOTCH1 (N1ICD) in the N1ICD-RBPJ-complex, as N1ICD-mediated transcriptional activity is reduced after overexpression of FHL1. Concomitantly, NOTCH1 target genes are also reduced. As NOTCH1 signaling is frequently overactive in BTCs while FHL1 expression is decreased, this suggests an imbalance between FHL1 binding to RBPJ and N1ICD binding to RBPJ, thereby resulting in expression of oncogenic target genes. Reconstitution of FHL1 leads to tumor suppression, which could partially be attributed to attenuated NOTCH1 signaling. After co immunoprecipitation of FHL1 and subsequent mass-spectrometric analysis of the associated proteins, additional transcription factors have been found, such as GTF2I. Thus, this study shows that FHL1 is a tumor suppressor in GBC and indicates that the miR 4502/FHL1 axis with subsequently affected genes, can be an important new pathway in order to develop a targeted therapy for GBC. This may in the long term improve the outcome of GBC patients.