Potential First Supervisor name: Dr Ildiko Gyory Affiliation

Potential First Supervisor name: Dr Ildiko Gyory
Affiliation: Department of Biochemistry, University of Leicester
Potential Second Supervisor name: Professor Catrin Pritchard
Affiliation: Department of Biochemistry, University of Leicester
PhD project title: Investigating the role of a novel tumour suppressor in an animal model of
K-RAS-driven lung cancer
University of Registration: Leicester
Project outline, timing, and budget
1. Project outline describing the scientific rationale of the project (max 4,000 characters incl.
spaces and returns)
Project outline
Cancer development is a multi-step process that transforms normal cells to a malignant
state. Several independent, sequential and stable events must occur to disturb regulation
before the tumour cells become invasive.
Oncogenic KRAS is one of the most prevalent tumour initiator in human lung
adenocarcinomas. Paradoxically, these RAS mutants inhibit proliferation because normal
cells respond to inappropriate proliferation signals by activating tumour suppressors. To
overcome this barrier of cell growth a key tumour suppressor must be inactivated.
The EBF family of DNA-binding proteins represents a new class of tumour suppressors
whose inactivation blocks normal development of diverse tissues resulting in human cancer.
The most-studied family member, Ebf1 is required for B lymphocyte development and its
tumour suppressor role in acute lymphoid leukaemia has been proven by animal
experiments and by genetic studies. Using a conditional knockout allele, our group has
shown that Ebf1 is required for replication and survival of B cells.
Genetic evidence suggests that Ebf1 is expressed in human lung tissue and expression
significantly decreases in a set of non-small-cell lung carcinomas. These and the proven
etiologic role of Ebf1 in lymphoid malignancies, have led us to the hypothesis that Ebf1 plays
an etiologic role in lung tumourigenesis.
The aim of this proposal is to study the role of Ebf1 in lung cancer pathogenesis in a unique
animal model that simultaneously activates oncogenic KRAS and inactivates the Ebf1
tumour suppressor.
In the LSL-KRASG12D mice, the endogenous Kras locus is targeted to introduce the
oncogenic G12D mutation, alongside with a conditionally removable floxed translational
STOP codon, ensuring that endogenous level of oncogenic KRAS G12D protein is
expressed following removal of the Stop element. Excision of the STOP signal is carried out
by a recombinant Adenovirus expressing the Cre recombinase (Ad-Cre), which allows
synchronised tumour initiation. The LSL-KRAS G12D-Ad-Cre mice develop atypical
adenomatous hyperplasia (AAH), which is a precursor to pulmonary adenocarcinoma both in
mice and in human patients.
In the LSL-KRASG12D-Ad-Cre mice accumulation of additional spontaneous mutations that
transform AAH to adenocarcinoma takes about 16 weeks. However, introduction of targeted
mutations in specific genes has been shown to result in accelerated tumour progression.
The Ebf1fl/fl mouse strain was shown to efficiently delete the Ebf1 gene by transgenic Cre
expression, and when crossed to the LSL-KRASG12D mice, the anticipated result is the
simultaneous KRAS activation and reduction (+/fl) or loss (fl/fl) of Ebf1 expression upon AdCre infection.
In the animal facility of the University of Leicester, we maintain both the LSL-KRASG12D and
the Ebf1fl/fl mice. The Pritchard lab is interested in how mutant proteins of the Ras signal
transduction pathway initiate and drive human cancer development, and the Gyory lab has
the complementing interest in transcriptional and epigenetic regulation, as two processes
that drive oncogenesis in concert with signalling.
Ebf1 has been shown to alter either the chromatin state or transcription of its target
loci during B lymphocyte development, but the role of Ebf1 in lung tissue or in
lung carcinogenesis has never been addressed before. We are planning to examine
the chromatin and transcription level changes that are associated with loss of Ebf1
expression during malignant transformation of lung epithelial cells in the proposed animal
model. The required animal costs are covered by an MRC grant to the Gyory lab, and we
expect that the mouse colony will be set up in advance. The statistical tumour progression
analysis will be done by the time the student arrives so she/he would start the analysis
of the relevant tissues at the molecular level. In the 1st year the focus will be
signalling and gene expression, followed by the analysis of epigenetic changes in the next
1.5 years.
Please list the techniques that will be undertaken during the project.
List of techniques to be used during the PhD project
Standard molecular techniques, transgenic mouse models, flow cytometry, fluorescence
microscopy, ChIP-sequencing, 4-thiouridine tagging and purification of nascent RNA and
qPCR to detect changes in gene expression

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