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Title: CRAF signalling in RAS mutant melanoma cells
Author: Jennifer, Sonja J.
ISNI:       0000 0004 2681 8900
Awarding Body: Institute of Cancer Research (University Of London)
Current Institution: Institute of Cancer Research
Date of Award: 2009
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The classical ERK/MAPK signalling pathway regulated proliferation, differentiation and survival. At the core of this pathway is the serine/threonine-specific protein kinase RAF, whose activation downstream of RAS leads to the sequential activation of the kinases MEK and ERK. There are three RAF proteins (ARAF, BRAF, CRAF) and three RAS proteins (HRAS, KRAS, NRAS) in humans. In melanoma, ERK is hyperactivated because BRAF is mutated in 44% and RAS is mutated in 24% of the cases. Importantly, downstream of oncogenic RAS, signalling to MEK and ERK occurs exclusively through CRAF. CRAF is regulated at the level of phosphorylation but until recently, controversy surrounded the identity of its upstream kinases. Here, a semi-high-throughput screen was developed in which NRAS mutated melanoma cells were screened using a human siRNA kinoma library in 96-well format. The rationale was to identify kinases that are required to activate CRAF using ERK phosphorylation as a readout. Primary hits were validated in secondary and tertiary screens by western blotting and finally by quantitative PCR to ensure knockdown of target proteins at the RNA level. On close examination, the majority of the hits were found to have their inactivating effects by down-regulating NRAS at both the level of protein and RNA rather than by depleting their specific targets. One kinase (casein kinase 2), which I had identified as a confirmed hit, was published during the course of this work. This study demonstrates how off-target effects can confound high-throughput siRNA screening campaigns. In melanoma cells with BRAF mutations, MEK, pan-RAF and BRAF inhibitors block ERK signalling. The second aspect of my project was to examine the effects of these inhibitors on signalling in cells with different genetic lesions. Intriguingly, I found that in cells which harbour oncogenic RAS, inhibition of BRAF drives BRAF into a complex with CRAF, resulting in BRAF hyperphosphorylation. Furthermore, under these conditions, CRAF is strongly activated, leading to an increase in ERK signalling. Notably, these effects are not seen when oncogenic BRAF is inhibited. When RAS mutant cells are treated with a pan-RAF inhibitor CRAF undergoes paradoxical activation whereby it is activated as a consequence of the drug binding to BRAF and complex formation, but is inhibited by direct binding of the pan-RAF inhibitor. Importantly, I also show that a kinase-dead mutant of BRAF constitutively binds to CRAF in RAS mutant cells and simulates ERK activation. Crucially, through collaboration with another student in the Marais laboratory, we show that the interaction I have elucidated causes accelerated tumourigenesis in mouse models. My data show that BRAF-selective inhibitors will only be effective in BRAF mutated cells and should not be administered to patients with RAS mutant tumours. This study demonstrates the importance of determining the underlying genetic cause of disease when selecting treatment options for patients.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available