German Title: Die Rolle anti-apoptotischer Bcl-2 Proteine für Entstehung und Progression des kolorektalen Karzinoms

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Abstract

Anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins such as Bcl-2 itself, Bcl-xL and Mcl-1, prevent mitochondrial activation and thereby the induction of the intrinsic apoptotic signaling pathway. Since the avoidance of cell death is a prerequisite for malignant transformation, anti-apoptotic proteins are frequently overexpressed in tumor cells. Additionally, there is growing evidence that Bcl-2, Bcl-xL and Mcl-1 also play a role in other cellular processes, such as cell cycle regulation, DNA repair and autophagy induction, which can also be important for the onset and progression of cancer. One of the most frequently diagnosed malignancies worldwide is colorectal cancer. Even though improvements in treatment and population screenings have led to a decreased mortality in many countries, especially patients with metastasized colorectal cancer still face a poor prognosis. Until now, it has been an open question whether and to which extend anti-apoptotic Bcl-2 proteins influence colorectal cancer initiation, progression and metastasation, with a few studies showing contradictory results. Hence, the purpose of this work was to shed more light on the role Bcl-2, Bcl-xL and Mcl-1 play for the maintenance of intestinal tissue homeostasis and for colorectal cancer development and outgrowth. First, their expression levels in the human intestinal mucosa as well as in adenoma and adenocarcinoma tissue were determined. This revealed a significant increase of Bcl-xL in the malignant state and an unaltered expression of Bcl-2. By contrast, Mcl-1 has been found to be significantly downregulated in colorectal cancer specimens. Results obtained in subsequent in vitro experiments clearly showed that Mcl-1 has an anti-proliferative effect which cancer cells preclude by downregulation of the protein. Neither in intestinal epithelial cells nor in colorectal cancer cells has a cell cycle inhibiting mode of action been described so far for Mcl-1. Additionally, further in vitro experiments have shown for the first time that the siRNA mediated silencing of anti-apoptotic Bcl-2 proteins significantly decreased the migratory capacity and invasiveness of human colorectal cancer cells. In a second step, protein functions were studied in further detail in vivo. Since previous publications showed that the constitutive deletion of both Bcl-xL and Mcl-1 induces embryonic lethality, two intestine-specific knockout mouse models were generated during this work. For the first time, they allowed to study functioning of Bcl-xL and Mcl-1 in the murine intestine. The mouse models revealed a strong discrepancy between Bcl-xL and Mcl-1, regarding their influence on tissue maintenance and tumorigenesis. While Bcl-xL turned out to be dispensable for normal tissue homeostasis, it has been found to be a crucial factor for colorectal cancer cell survival in a chemically induced tumor model. This confers Bcl-xL tumor-promoting properties and explains its overexpression in human adenomas and adenocarcinomas. The loss of Mcl-1, by contrast, caused a severe intestinal phenotype, comprising high levels of cell death, an accompanying increase of proliferation and chronic inflammation. From an age of about six months, spontaneous tumorigenesis was observed in intestine-specific Mcl-1 knockout mice which was promoted by the loss of the anti-proliferative effect Mcl-1 exerts on intestinal epithelial cells and the inflammatory environment. Therefore, it has been proven that Mcl-1 possesses tumor-suppressing properties in the intestine, what explains its downregulation in human colorectal cancer specimens. The presented results highly recommend the utilization of Mcl-1 sparing inhibitors in the context of colorectal cancer treatment. A first step towards clinical application was done in this work by treating viable human colorectal cancer tissue ex vivo with the Bcl-xL/Bcl-2-specific inhibitor ABT-737. Subsequent analyses revealed a significantly decreased viability of human colorectal cells in presence of the inhibitor. Since proliferation turned out to be unaltered under ABT-737 treatment, inhibition of Bcl-xL in combination with classical chemotherapy could be an interesting approach for further studies with a focus on clinical applicability.