Design and synthesis of molecular inhibitors of cFLIP activity as a therapeutic strategy to target breast cancer cells and cancer stem-like cells

Hayward, Olivia A 2016. Design and synthesis of molecular inhibitors of cFLIP activity as a therapeutic strategy to target breast cancer cells and cancer stem-like cells. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) is a known anti-cancer agent which exhibits cancer cell specificity. Its efficacy in breast cancer is limited, however, as illustrated by the resistance demonstrated in a number of cell lines. Cellular FLICE-Like Inhibitory Protein (c-FLIP) has been demonstrated to play a key role in TRAIL resistance in these cell line models, through its competitive interference with caspase-8 recruitment to the death inducing signalling complex (DISC). Our lab has demonstrated that breast cancer 'stem cells', the cells proposed to be responsible for disease recurrence and metastasis, can be eliminated by combination of c-FLIP suppression, by siRNA, and TRAIL treatment. While it has been proposed that suppression of c-FLIP could improve TRAIL efficacy in breast cancer, there are no pharmacological c-FLIP specific inhibitors available. The purpose of this study therefore was to design and validate novel pharmacological inhibitors of c-FLIP and to determine their tumour efficacy in models of breast cancer. Using in silico modelling, we have modelled the structure of c-FLIP and associated proteins, and, using virtual pharmacophore screening, we have identified 19 potential small molecule c-FLIP inhibitors. In vitro cell viability assays demonstrated 3 of these inhibitors to sensitise TRAIL resistant MCF-7 and BT474 breast cancer cell lines to TRAIL (p<0.05) at micromolar concentrations, with several additional compounds showing a partial ability to sensitise to TRAIL. Each of the three lead inhibitors were shown to be acting via caspase-8 and therefore in a TRAIL specific manner. Two of the lead compounds in combination with TRAIL significantly reduced tumoursphere forming potential in an in vitro assay which represents breast cancer stem cell like traits. The ability of our primary compound to target cells with breast CSC-like traits was further supported using a colony forming assay, which demonstrated a significant reduction in the potential for clonal expansion of breast cancer cells following treatment with the novel agent in combination with TRAIL. Acceptor-photobleaching FRET was successfully utilised to show the mechanism of action of our lead inhibitors to indeed be based on interfering with the c-FLIP FADD interaction. TALEN-mediated targeting of c-FLIP is being optimised to allow for further evaluation of these agents in addition to performing mutagenesis studies in a c-FLIP null background to further delineate the role and mechanism of c-FLIP in the sensitization of breast cancer stem cells to TRAIL. Here we have identified several small molecule c-FLIP inhibitors which are able to specifically sensitise resistant breast cancer cells to TRAIL induced apoptosis via interfering with c-FLIP FADD interaction at the DISC, and thus allowing caspase-8 mediated apoptosis to occur. These early results show promise for our lead compounds as potential therapeutics in breast cancer, via sensitisation to TRAIL.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Biosciences Pharmacy
Subjects:	R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Funders:	Cancer Research Wales
Date of First Compliant Deposit:	30 March 2016
Last Modified:	08 Jul 2021 10:56
URI:	https://orca.cardiff.ac.uk/id/eprint/86899

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