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

Ovarialkarzinome gehören zu den weitverbreitetsten und lebensgefährlichsten gynäkologischen Tumoren mit bislang begrenzten Behandlungsmöglichkeiten. Ein besseres Verständnis der genetischen Änderungen in der Pathogenese des Ovarialkarzinoms ist von entscheidender Bedeutung. Im Rahmen dieser Doktorar...

Full description

Saved in:
Bibliographic Details
Main Author: Abuharbeid Shaker
Contributors: Czubayko, Frank (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!

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.