Catrin Pritchard - Overview
Disregulation of the Ras/Raf/MEK/ERK pathway plays
a major role in cancer pathogenesis and agents that target components
of this pathway are proving to be important compounds for new cancer therapies.
In collaboration with Dr Richard Marais (Institute of Cancer Research,
London), we are using transgenic and gene targeting technology to fully
understand the regulation and functions of one component of this pathway:
the Raf family of protein kinases. The Raf family comprises three members
in mammals: A-Raf, Raf-1 and B-Raf. We have derived fibroblast cell lines
that contain individual knockout mutations of each raf gene as well as
cell lines with a knockin mutation of Y340FY341F in Raf-1. These cells
are being used to characterise the individual contribution of each Raf
isotype to the Ras/Raf/MEK/ERK pathway as well as their roles in the control
of cell proliferation, apoptosis, cell shape and motility, oncogenic transformation
and tumourigenesis.
A-raf knockout cells proliferate normally, show no changes in cell shape,
motility, apoptosis or Ras transformation and ERK activation is normal
in these cells. Cell lines with a knockout mutation of Raf-1 have no changes
in ERK activation in response to a wide variety of extracellular stimuli.
They also proliferate normally and have no changes in cell shape or cell
motility, yet they have increased susceptibility to several apoptotic
agents. This role of Raf-1 in apoptosis suppression surprisingly appears
to be independent of the accepted role of Raf-1 in activating MEK/ERKs
since a MEK kinase inactive version of Raf-1, containing the Y340FY341F
mutation, fully rescues the apoptosis phenotype. By contrast, B-raf knockout
cells have a complex spectrum of abnormalities that arise as a result
of a severe disruption to ERK activation. These data provide strong evidence
that the A-Raf and Raf-1 isotypes may not participate in the Ras/Raf/MEK/ERK
cascade in vivo. The main function of Raf-1 is in suppression of apoptosis
and this function appears to be independent of ERK activation. B-Raf appears
to be the main, if not the only Raf protein participating in the Ras/Raf/MEK/ERK
signalling cascade in vivo. These results have important implications
for the development of cancer therapies targeted against the Ras/Raf interaction.
We are continuing to use gene targeting technology to investigate the
MEK kinase independent functions of Raf-1 and A-Raf, and particularly
the role of Raf-1 in apoptosis suppression. We are also focussing on the
role of B-Raf as a critical signalling intermediate between Ras GTPases
and the ERKs and its role in the manifestation of the tumour phenotype.
In order to facilitate this work we have established an Embryonic Stem
cell facility at Leicester which works in conjunction with the transgenic
unit to undertake all gene targeting/transgenic projects for our own group
as well as for external contracts.
