The Fibroblast Growth Factor-binding Protein (FGF-BP) and the Human Epidermal Growth Factor Receptor-2 (HER-2): functional studies on two gene products relevant in Ovarian cancer

Ovarian cancer is one of the most widespread and lethal gynecological malignancies with so far limited treatment options. A better understanding of the genetic changes in ovarian pathogenesis is of critical importance. In this thesis, two gene products were studied: the fibroblast growth factor-binding protein (FGF-BP) and the human epidermal growth factor receptor-2 (HER-2). FGF-BP is a heparin-binding protein which interacts with FGFs releasing them from the extracellular matrix and hence playing a significant role in extracellular FGF bioactivation. In this work, the immunohistochemical analysis of tissue microarrays (TMAs) revealed for the first time FGF-BP overexpression in 40% of invasive ovarian carcinomas. Since none of the normal ovarian tissues showed similar FGF-BP immunopositivity, these data suggest that FGF-BP overexpression may represent an acquired malignant phenotypic feature of ovarian carcinoma. To further explore the molecular mechanism of FGF-BP action, confocal microscopy was employed. Dependent on the cell line, FGF-BP was either localized in the cytoplasm and released through a classic secretion pathway employing ERGIC or, despite a classical signal peptide, showed a nuclear localization. Interestingly, upon coexpression with nuclear FGF-2, cytoplasmic FGF-BP was translocated and colocalized with FGF-2 in the nucleus. Additionally, the dependence of the cellular uptake of exogenous FGF-BP on the expression of and interaction with FGF-2 was demonstrated. Various truncated FGF-BP mutant constructs showed that extensive truncations at the C-terminal end of FGF-BP had no effect on the subcellular localization and FGF-2 interaction, while upon N-terminal truncations nuclear colocalization and interaction of FGF-BP with FGF-2 was lost. Proliferation assays provided evidence that biological effects of FGF-BP depend on the cell line. While in SW-13 adrenal carcinoma cells the stable transfection of FGF-BP induced cell proliferation, inhibitory activity of FGF-BP in COS-7 cells was demonstrated. Moreover, FGF-BP-mediated stimulation of colony formation in soft agar was lost upon N-terminal truncations of FGF-BP in SW-13 cells, whereas C- or N-terminal truncations abolished the inhibitory effect of FGF-BP on COS-7 cell proliferation. The induction of cell proliferation resulting from the expression of FGF-2 in COS-7 cells, which also express FGF receptors, was lost upon endogenous expression or exogenous addition of FGF-BP. Hence, in addition to its already published extracellular role, FGF-BP exerts intracellular, nuclear functions. More specifically, dependent on the cell line FGF-BP displays inhibitory or stimulating activities upon interaction with FGF-2 in the nucleus. HER-2 belongs to the epidermal growth factor (EGF) receptor family and plays an important role in human tumors. However, clinical and experimental data indicating effects of HER-2 overexpression on tumor cell sensitivity towards chemotherapy are conflicting as to whether elevated HER-2 levels lead to increased resistance or higher sensitivity of tumors. This is particularly true for ovarian carcinomas and ovarian carcinoma cell lines, where HER-2 overexpression has been found in considerable percentages. In this work, the role of HER-2 expression and signaling levels pertaining to paclitaxel and rViscumin chemoresistance were explored. Treatment of SKOV-3 cells with two newly developed low molecular weight inhibitors of HER-2 tyrosine kinase activity resulted in a decrease in the cellular paclitaxel sensitivity. These data confirmed previous data regarding treatment with the HER-2 inhibitory antibody trastuzumab (Herceptin), which is well established in tumor therapy. Using various isogenic SKOV-3 cell lines with ribozyme-mediated stable reduction of HER-2 expression levels, a ‘HER-2 gene dose effect’ of paclitaxel cytotoxicity was established, while doxorubicin and cisplatin cytotoxicity remained unchanged. To elucidate the underlying mechanisms of this effect, paclitaxel- or HER-2-mediated alterations in phosphorylation of MAP kinase p42/44, stress-activated protein kinase/Jun-terminal kinase (SAPK/JNK), and p38, and effects on the activation of caspase-3, caspase-7, and bcl-2 were analyzed. Activation of MAP kinases was dependent on HER-2 expression levels but did not change upon paclitaxel treatment. Paclitaxel-induced bcl-2 phosphorylation and hyperphosphorylation was independent of HER-2 expression levels. Finally, it was shown that paclitaxel utilizes a caspase-independent pathway of induction of apoptosis in SKOV-3 ovarian carcinoma cells. The selective depletion of HER-2 by ribozyme-targeting also decreased the cellular sensitivity of SKOV-3 cells towards the recombinant cytostatic mistletoe rViscumin, establishing a ‘HER-2 gene dose’ dependence of rViscumin cytotoxicity, which is not mediated by altered cellular rViscumin binding/internalization. The analysis of the underlying molecular effects of rViscumin treatment demonstrated that members of the MAPK family, p42/44, SAPK/JNK, and p38 are activated in a HER-2 and rViscumin concentration-dependent manner. While no rViscumin-mediated activation of caspase-3 and caspase-7 were observed, HER-2-dependent downregulation of the anti-apoptotic molecule bcl-2 was demonstrated. In conclusion, by focusing on two cancer-relevant genes, this work establishes FGF-BP as a new potential target molecule in ovarian cancer therapy and explores its intracellular mechanism of action. Furthermore, this thesis provides new insights into the role of HER-2 in ovarian cancer sensitivity towards cytostatic drugs like paclitaxel or rViscumin which may allow to better assess the efficacy of both drugs in a clinical setting.