Charakterisierung von MUC16 (CA125) Knockdown Varianten einer humanen Ovarialkarzinomzellinie

Das tumorassoziierte Antigen CA125 (MUC16) wird häufig von Ovarialkarzinomen aber auch anderen epithelialen Tumoren überexprimiert. Obwohl CA125 Serumspiegel häufig zur Verlaufskontrolle von Ovarialkarzinompatientinnen angewendet werden, ist die physi-ologische Funktion dieses Muzins weitgehend unbe...

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Bibliographic Details
Main Author: Kage, Sophie
Contributors: Wagner, Uwe (Prof. Dr. med. ) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
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Table of Contents: The tumor associated antigen CA125 (MUC16) is commonly highly expressed in ovarian cancer and can also be detected in other tumour of epithelial origin. Although CA125 se-rum levels are frequently used to monitor patients with ovarian cancer, its physiological role is largely unkown. The aim of this study was to investigate the impact of MUC16 gene silencing on the growth properties of ovarian cancer cells. Therefore the cellular effects linked to oncogenesis, such as proliferation, cell cycle and apoptosis after transient and stable transfection with MUC16 short hairpin RNA (shRNA) in the ovarian cancer cell line OAW-42H8+ were analysed. Furthermore, alterations in cell adhesion, migration, and in-vasion were evaluated in stable MUC16 knockdown clones. First of all, the results showed a significant suppression of growth by induction of caspase-dependent apoptosis after transient transfection with MUC16 shRNA. In these cells Caspase 3 was found to be activated and was associated with increased cleavage of PARP. Growth inhibition and increased apoptosis could be confirmed in stable MUC16 knock-down clones, although caspase-dependent death pathways seemed no longer to be activat-ed. Neither Caspase 3 activation nor PARP cleavage were detected in stable knockdown clones. Therefore these clones were analysed for mitochondrial release of caspase-independent cell death effectors. Indeed, translocation of AIF from mitochondria to the nucleus was observed in the stable MUC16 knockdown clone. The data indicate a shift from caspase-dependent apoptosis to caspase-independent cell death in stable long-term surviving MUC16 knockdown clones in ovarian cancer cell line OAW-42H8+. Besides inhibition of proliferation through induction of apoptosis a significantly reduced clonogenicity was observed after stable MUC16 knockdown. As the PI3K/AKT signaling pathway is often activated in ovarian cancer cells and is additionally involved in multiple cell functions like growth, cell cycle control and apoptosis, the influence of MUC16 on this pathway was further evaluated. AKT activation as well as AKT expression levels were suppressed in stable MUC16 knockdown clones. Furthermore downstream effectors of AKT like 4EBP1 and S6, regulating protein translation and therefore tumor cell growth, were shown to be inhibited in MUC16 knockdown clones. Additionally the reduced AKT activation was linked to suppression of anti-apoptotic Bcl-2 in these cells. The data pre-sented here provide further evidence for MUC16-mediated regulation of tumour growth through the PI3K/AKT pathway, although other signaling pathways may be involved. In contrast to cell growth and apoptosis MUC16 was shown to have no effect on migration and invasion in the OAW-42H8+ cell line. These cells in generally displayed a non-motile and non-invasive phenotype which was not altered by MUC16 knockdown. Our results provide evidence for a central role of MUC16 in ovarian cancer cell survival pathways. Thereby different apoptosis pathways as well as the PI3K/AKT signaling path-way are influenced by MUC16 Knockdown. MUC16 is therefore a promising candidate for targeted cancer therapy. But to develop further therapy options a better understanding of its physiological function and interaction with signaling molecules in ovarian cancer cells in vivo and in vitro is necessary.