Modulation und funktionelle Relevanz des RANKL/TRAIL/Osteoprotegerin-Systems beiMammakarzinomzellen

6. Zusammenfassung Obwohl es in der Behandlung des Mammakarzinoms deutliche Fortschritte gibt, bleiben die Therapieoptionen für Hormonrezeptor-negative und ossär metastasierte Tumoren begrenzt. Die Entdeckung des RANKL/RANK/OPG-Systems im Jahre 1997 hat das Verständnis über die komplexe Regulatio...

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Bibliographic Details
Main Author: Rachner, Tilman Daniel
Contributors: Hofbauer, Lorenz (Prof.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2009
Online Access:PDF Full Text
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Summary Despite advances in the therapy of breast cancer, treatment options for women suffering from hormone receptor-negative breast cancer or bone metastases remain limited. The discovery of the RANKL/RANK/OPG system in 1997 has fundamentally influenced our knowledge about the complex regulation of bone metabolism. The cytokine RANKL, its receptor RANK and the decoy receptor OPG are essential regulators of osteoclast biology. OPG is also thought to play a role in tumor biology. It was shown that RANKL enhances the migration of breast and prostate cancer cells in vitro and in vivo. TRAIL is a member of the TNF ligand family that selectively induces apoptosis in malignant cells. As a decoy receptor for both RANKL and TRAIL, OPG has the unique ability to regulate two fundamental aspects of tumor biology, apoptosis and migration. Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. In the first part of this study, the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231 were analyzed. In MCF-7 cells, which predominantly express ER-, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively. Furthermore, both cell lines expressed RANKL. While IL-1β induced RANKL expression in both cell lines, 17β-estradiol decreased RANKL expression only in MCF-7 cells. The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. This indicates that androgens indirectly influence OPG expression in breast cancer cells after aromatization and binding of the ER. In the second part of this study the expression and modulation of OPG and TRAIL by IL-1β and dexamethasone were analyzed in MCF-7 and MDA-MB-231 cells. In both cells, OPG mRNA levels and protein secretion were dose- and time-dependently enhanced by IL-1β and suppressed by dexamethasone. In contrast to MCF-7 cells, MDA-MB-231 abundantly expressed TRAIL mRNA, which was enhanced by IL-1β and inhibited by dexamethasone. TRAIL activated pro-apoptotic caspase-3, -7, and poly-ADP-ribose polymerase and decreased cell numbers of MDA-MB-231, but had no effect on MCF-7 cells. Gene silencing using siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non-target siRNA-treated MDA-MB-231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG and these protective effects were reversed by blocking OPG with its specific ligand RANKL. The association between cancer cell survival and OPG production by MDA-MB-231 cells was further supported by the finding that modulation of OPG secretion using IL-1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively. Bisphosphonates are established drugs in the treatment of bone metastasis that inhibit osteoclast activity and interrupt the vicious cycle of osteoclast-tumor cell interactions. In the third part of this study the direct effects of zoledronic acid on MDA-MB-231 and MCF-7 breast cancer cells were evaluated. While zoledronic acid (100 µM) inhibited MDA-MB-231 cell proliferation after 72 h, and induced apoptosis via activation of caspase-3 and -7, it had only minor effects in MCF-7 cells. In addition, zoledronic acid induced the expression of TRAIL in MDA-MB-231 cells, but had no effect on the expression of its decoy receptor OPG. In MCF-7 cells, both cytokines were suppressed by zoledronic acid. In summary, this study investigated two potential concepts to target breast cancer. First, the direct apoptotic effects of zoledronic acid and its underlying mechanisms on the ER negative MDA-MB-231 cells were demonstrated. Second, the regulation of the OPG/TRAIL/RANKL system by multiple disease relevant substances was analyzed. The role of endogenously secreted OPG as a survival factor for TRAIL sensitive breast cancer cells was demonstrated as well as the possibility to therapeutically target this ability by pharmacologically suppressing the OPG secretion.