Browsing by Subject "Protein p53"

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    Charakterisierung primärer Tumor-assoziierter Fibroblasten und Tumorzellen aus bronchialen Karzinomen und Untersuchung ihrer Reaktion auf zielgerichtete und zytotoxische Therapie
    (2013) Schmid, Jens Oliver; Aulitzky, Walter E.
    Worldwide lung cancer is the leading cause of death among all malignancies. This is largely due to its high frequency of diagnosis and its poor 5-year survival rate of 15%. As a solid tumor, lung cancer consists of tumor cells and a variable stromal part, that is made up of a cellular and a non-cellular fraction. The stroma influences several processes like growth, invasion of surrounding tissues, metastatic spread as well as tumor supply of oxygen and nutrients. Thereby, the stroma is dominated by the cancer-associated fibroblasts (CAFs), actively shaping the microenvironment through the secretion of soluble factors and the synthesis of extracellular matrix (ECM) components. While there are several studies with the aim of identifying the differences between CAFs and normal fibroblasts (NAFs) of the breast, there is only little information about those differences in the corresponding cells of the lung. One of the aims of the study was the identification of the molecular differences between CAFs and NAFs derived from lung tissue. A further objective was the investigation of therapy effects under conditions that mimic the situation in vivo. Therefore, an ex vivo-model allowing the culture of primary lung tumor tissue had to be evaluated. Afterwards, the effect of the epidermal growth factor receptor (EGFR) inhibitor Erlotinib on tumor cell proliferation was investigated in this model system. To investigate a response of both, tumor cells and their adjacent CAFs to chemotherapy, lung cancer tissue samples were treated with cisplatin. Finally, owing to their important role in tumors, CAFs were chosen as a target for therapy using small molecule inhibitors, with the aim of inhibiting their stimulatory effect. Molecular comparison of isolated CAFs and the corresponding NAFs of 9 lung cancer patients revealed a significantly different expression of 60 genes. The identification of a set of differentially regulated genes is quite surprising because of the assumable activation of NAFs due to culture conditions. This indicates that CAFs are more than just activated fibroblasts, which are found at sites of tissue injury. Rather, they are a distinct cell type showing parallels to activated fibroblasts. Expression data for 46 of the 60 identified genes were available in a Non-Small Cell Lung Cancer collective comprising of 342 patients. As it turned out, a NAF-like expression of the genes was associated with a significantly better survival prognosis. Another central objective of the work was the investigation of the tumor cell response to therapy in an intact tissue. An already established tissue culture system required initial validation. This was done by comparing the tissue which has been cultivated for 4 days with the corresponding tissue, that has been fixed immediately after surgery. No significant changes in morphology and biological function were detected. Thus the model system adequately mimics the situation in the patient and a negative effect of the culture could be excluded. This makes the system an excellent opportunity to investigate the effect of a drug under in vivo-like conditions. Treatment of tissue samples, characterized for EGFR expression, and EGFR, and KRAS gene status, with the small-molecule inhibitor Erlotinib displayed no effect on the proliferation of the analysed tumor cells.This reflects the situation in the clinics quite adequately where only a small proportion of patients benefits from the treatment with the EGFR-inhibitor Erlotinib. To follow-up, the reaction of CAFs and tumor cells on a chemotherapeutical treatment was investigated under in vivo-like conditions. Interestingly, cisplatin led to a parallel accumulation of p53 and induction of cell death in tumor cells and their adjacent CAFs. Thereby, the p53 accumulation of CAFs seems to be dictated by their tumor cells because the same CAFs which do not accumulate p53 in the tissue, respond to cisplatin with the accumulation of p53 in the isolated state. Therefore, it is tempting to speculate that tumor cells modulate the DNA damage response of their microenvironment, with the objective to raise their own chemoresistance. Inhibiting CAF proliferation was examined as a feasible approach to inhibit their tumor-stimulating properties. Screening of a kinase inhibitor library consisting of 160 small molecule inhibitors resulted in the identification of PDGFR signaling as a promising target. Among the FDA approved PDGFR inhibitors Dasatinib turned out to be the most potent inhibitor of CAF proliferation, resulting in a molecular phenotype comparable to that of normal fibroblasts. Furthermore, the Dasatinib-mediated changes in CAFs led to the secretion of factors, inhibiting the proliferation of lung tumor cells. In contrast, the secreted factors of untreated CAFs stimulated their proliferation. Together, these results indicate that Dasatinib treatment is a promising approach to reduce the tumor promoting capacity of CAFs.