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Title: Resistance mechanisms to mTOR inhibition in renal cancer
Author: Earwaker, Philip L.
ISNI:       0000 0004 6062 6160
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Introduction: Advanced clear cell renal cell cancer (CCRCC) is incurable, but molecularly targeted treatments have improved the prognosis. One such molecular target is the mammalian target of rapamycin (mTOR) and two rapalogues (everolimus and temsirolimus) have been licensed for the treatment of advanced CCRCC, but resistance to treatment is a significant problem. Novel mTOR kinase inhibitors show more complete blockade of mTOR downstream targets, and greater in vitro anti proliferative activity, although resistance remains a problem. Hypotheses: 1) CCRCC cell lines, resistant to mTOR kinase inhibition could be generated in vitro through continuous culture in drug, and 2) mediators of resistance could be identified through screening cells at the mRNA and protein/phosphoprotein level, or by examining the signalling in resistant cells to identify the re-activation of key mTOR targets. Methods: Resistant cells were created by continuous culture in the PI3K-mTOR kinase inhibitor BEZ235. Resistance was assessed by CellTiter-Glo viability assays and clonogenic assays. mRNA microarray and antibody arrays were conducted. Intra-cellular signalling was assessed by western blotting. The functional relevance of identified markers of resistance were assessed with small molecule inhibitors and siRNA protein depletion. Results: BEZ235 resistant cells were created and had a 14 fold higher growth inhibitory 50 (GI50) concentration compared to sensitive cells (99 nM vs. 7 nM). Array screening of the cells identified markers, but not mediators of resistance (MET, Abl, MEK/ERK and the Notch pathway). In BEZ235 resistant cells, the downstream mTOR target 4E-BP1 was rephosphorylated, despite evidence of on-going blockade of another mTOR target, S6. Recovery of 4E-BP1 phosphorylation was associated with increased protein expression of the mTOR binding partner RAPTOR, and could be reduced by depletion of RAPTOR, or by the addition of rapamycin. Both of these interventions partially reversed BEZ235 resistance. Conclusions: RAPTOR protein up-regulation, represents a novel mechanism of resistance to mTOR kinase inhibition and can be partially overcome by rapamycin. The combination of BEZ235 and rapamycin warrants further investigation to evaluate its potential to overcome resistance to mTOR kinase inhibition in RCC.
Supervisor: Macaulay, Valentine Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available