Exploring mTOR signalling as a targeted approach against ovarian cancer

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonOvarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer suffers from a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH-2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. We demonstrate that MDAH-2774 cells have higher proliferative capacity than SKOV3 cells, suggesting different inherent signalling capacities. Using a wound healing assay we show that inhibition of the mTOR pathway using Rapamycin, rapalogues, Resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 hours in these cell lines. We extended these findings up to 72 hours with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 or 3 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer. We also show that increased DEPTOR is a positive prognostic predictor in ovarian cancer and is decreased in later stage disease. Finally, we demonstrate that DEPTOR upregulation is mirrored in RNA extracted from whole blood of ovarian cancer patients. This in conjunction with the development of ultra-fast qPCR instrumentation can provide a valuable point of care testing platform for non-invasive diagnostics. A screening method that could provide early detection of ovarian cancer would represent a sea change in the diagnosis and treatment of this commonly lethal disease

Ex-vivo signal transduction studies in chronic lymphocytic leukemia

Microenvironmental signaling is pivotal to chronic lymphocytic leukemia (CLL) pathology; therefore understanding how to investigate this pathway by both protein and chemical methods is crucial if we are to investigate and correlate biological changes with therapeutic responses in patients. Herein, we describe the use of western blotting also referred to as immunoblotting as a method that can semiquantitatively evaluate changes in protein expression following receptor engagement; this includes B cell receptor (BCR) signaling following stimulation with anti-IgM (Blunt et al. Clin Cancer Res 23(9):2313–2324, 2017). It is important to note that immunoblotting should always be combined with other quantitative methods such as flow cytometry to confirm activation of these signaling pathways (Aguilar-Hernandez et al. Blood 127(24):3015–3025, 2016).</p

B-cell receptor signaling induces proteasomal degradation of PDCD4 via MEK1/2 and mTORC1 in malignant B cells

B-cell receptor (BCR) signaling plays a major role in the pathogenesis of B-cell malignancies and is an established target for therapy, including in chronic lymphocytic leukemia cells (CLL), the most common B-cell malignancy. We previously demonstrated that activation of BCR signaling in primary CLL cells downregulated expression of PDCD4, an inhibitor of the translational initiation factor eIF4A and a potential tumor suppressor in lymphoma. Regulation of the PDCD4/eIF4A axis appeared to be important for expression of the MYC oncoprotein as MYC mRNA translation was increased following BCR stimulation and MYC protein induction was repressed by pharmacological inhibition of eIF4A. Here we show that MYC expression is also associated with PDCD4 down-regulation in CLL cells in vivo and characterize the signaling pathways that mediate BCR-induced PDCD4 down-regulation in CLL and lymphoma cells. PDCD4 downregulation was mediated by proteasomal degradation as it was inhibited by proteasome inhibitors in both primary CLL cells and B-lymphoma cell lines. In lymphoma cells, PDCD4 degradation was predominantly dependent on signaling via the AKT pathway. By contrast, in CLL cells, both ERK and AKT pathways contributed to PDCD4 down-regulation and dual inhibition using ibrutinib with either MEK1/2 or mTORC1 inhibition was required to fully reverse PDCD4 down-regulation. Consistent with this, dual inhibition of BTK with MEK1/2 or mTORC1 resulted in the strongest inhibition of BCR-induced MYC expression. This study provides important new insight into the regulation of mRNA translation in B-cell malignancies and a rationale for combinations of kinase inhibitors to target translation control and MYC expression

Network analysis reveals a major role for 14q32 cluster miRNAs in determining transcriptional differences between IGHV-mutated and unmutated CLL

Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells’ innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with the MEG3 lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3′UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation

BCR signaling contributes to autophagy regulation in chronic lymphocytic leukemia

No abstract available