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Molecular mechanisms in the pathogenesis of granulosa cell tumours of the ovary
thesisposted on 2017-01-17, 00:24 authored by Jamieson, Stacey
Granulosa cell tumours of the ovary (GCT) represent a specific subset of malignant ovarian tumours. Two distinct subtypes have been described based on clinical presentation and histological characteristics, the juvenile and the adult form. Although GCT are generally thought to have a better prognosis than epithelial tumours, approximately 80% of patients with advanced stage or recurrent tumours succumb to their disease. Despite the impact of this disease on affected women, remarkably little is known about the molecular changes that give rise to GCT. The fundamental aim of the work presented in this thesis was to identify the genes which contribute through mutation or altered expression to the pathogenesis of GCT. GCT exhibit a molecular profile that is consistent with FSH-responsive granulosa cells of the late preovulatoy follicle. Two human GCT-derived cell lines, COV434 and KGN, have proven to be useful in vitro model systems to investigate this disease. Previous work in our laboratory revealed that the both the NF-κB and AP-1 transcription factors were constitutively activated in these cell lines. The primary focus of these studies was to establish the basis and pathogenetic significance of the NF-κB signalling pathway in the COV434 and KGN cell lines. Using selective chemical inhibitors, candidate components within the NF-κB pathway were targeted. The functional consequence of NF-κB pathway inhibition was the loss of cellular properties commonly associated with the progression of malignancy, that is, a dose dependent decrease in cell proliferation and cell viability and an increase in apoptosis. Constitutive NF-κB signalling appeared to occur downstream of IRAK-1 and RIP1 and upstream IκBα phosphorylation in the COV434 and KGN cell lines. The cell lines’ unexpected lack of response to two independent inhibitors of IKKβ, a catalytically active subunit of the IKK complex, implicated a role for this molecule in aberrant pathway activation, however, no evidence of mutations and/or altered expression of the IKKβ gene were observed in the cell lines. Whilst a non-conserved single nucleotide polymorphism, reported to occur infrequently in certain Asian populations, was identified in the KGN cells, preliminary experiments utilising an IKKβ null cell line suggest that the polymorphism does not contribute to constitutive NF-κB activity, or resistance to the IKKβ specific chemical inhibitors. Small-molecule tyrosine kinase inhibitors (TKI) already in clinical use to treat cancers also proved useful tools to investigate the mechanisms of aberrant pathway activation. These studies demonstrated that the TKI, sorafenib, may be an effective therapeutic in the clinical treatment of advanced stage GCT and also implicated a role for BRAF in the constitutive activation of AP-1 via the classical MAPK/ERK pathway. The frequent presence of the FOXL2 C134W mutation in adult GCT was confirmed, and was found not to be associated with altered FOXL2 expression. Despite many clinical and molecular similarities, juvenile GCT lack the mutation and therefore clearly have a distinct molecular aetiology. The presence of the mutation in the KGN cell line and absence in the COV434 cell line indicate that they may be useful tools to study adult and juvenile GCT, respectively. The work in this thesis furthers our understanding of the role constitutively activated NF-κB and AP-1 transcription factors play in the molecular pathogenesis of GCT, and has identified a potential therapeutic agent that may be useful in the clinical management of patients with GCT.