Institute of Cancer School PhD Project Proposal
First Supervisor
Helen Hurst
Second supervisor
Louise Jones
Centre
Tumour Biology
Project Title
The role of Mage proteins in tamoxifen resistance
Project outline including background, aims and methodology (1 page only minimum font size 10)
Background:
The majority of breast tumours (70%) are oestrogen receptor positive (ER+) and can be treated with
tamoxifen, however, a significant proportion of patients will relapse with resistant disease. Newer
endocrine therapies have been developed, but meta-analysis of comparative clinical trials suggests that
switching protocols, whereby patients receive tamoxifen for 2-3 years before moving to an aromatase
inhibitor, offer the best long-term outcomes. Thus, research into the mechanisms behind tamoxifen
resistance (TR) remains highly relevant.
Expression profiling analysis of wt and TR lines identified MAGEA2 as a significantly upregulated gene.
This has been functionaly validated: silencing MAGEA2 in a TR line restores sensitivity to tamoxifen;
overexpression of MAGEA2 in ER+ lines confers resistance in vitro and as xenografts in nude mice;
MageA expression in clinical samples correlated with a poorer outcome on tamoxifen. Furthermore,
MageA2 can form complexes with p53 and inhibit its activation in tamoxifen media.
MAGEA2, a member of the class 1 MAGE gene family of cancer-testis antigens, defined by their lack of
expression in somatic adult tissues but frequent aberrant expression in tumours. The proteins are
characterised by a ~200 amino acid c-terminal MAGE-homology domain (MHD) whose precise function
is uncertain although it appears to act as a scaffold for protein-protein interactions. Tumour expression
of Mage proteins can elicit spontaneous immunogenicity in patients and immunotherapy strategies to
boost these responses, particularly for MAGEA3, are currently undergoing promising clinical trials.
Aims:
Our data suggests strongly that MAGEA2 overexpression is a determinant, and not merely a correlate,
of tamoxifen resistance and acts both in vitro and in vivo to inhibit growth arrest pathways normally
induced by tamoxifen. Due to the similarity between class 1 MAGE genes, we hypothesise that most
MageA and possibly B and C proteins may act in a similar manner to confer resistance. Project aims are
to determine to what extent other class 1 Mage proteins also contribute to resistance and to generate a
molecular understanding of the pathways altered by MAGE expression in ER+ breast epithelia. The
findings will be used to inform further translational/pre-clinical studies to target MAGE, or the pathways
they activate, in conjunction with tamoxifen treatment to improve patient outcomes and establish the
utility of Mage expression as a marker of de novo or acquired tamoxifen resistance in breast cancer.
Methodology:
1. Additional MAGE cDNA clones (IMAGE) will be stably overexpressed in ER+ breast tumour lines and
pools of clones will be tested for growth in tamoxifen as for MAGEA2. Tagged versions of MAGEA2 will
be checked for functional equivalence with wt protein, including co-immunoprecipitation (CoIP) with
p53 for aim 3. As a surrogate readout of p53 activity, we will use luciferase reporter assays; dose
escalation studies with MAGEA2 show progressive repression of p53-dependent reporter genes.
2. Further coIP assays will be used to explore interaction with additional pathways implicated in TR
including ER and its co-regulators. MageA2 expression confers resistance to a range of tamoxifen
concentrations but not to faslodex, suggesting that Mage proteins also act on the ER. We have also
found that the addition of tamoxifen stabilises and re-localises MageA2 to the nuclear compartment. We
will create a series of MAGEA2 deletion mutants to investigate which domain is required for its nuclear
localisation and its tamoxifen resistant function and determine if this correlates with the ability to
downregulate the p53 pathway.
3. Candidate gene approaches, however, do not always lead to new biological insights. We aim to
perform a screen to identify additional MageA2-interacting proteins using a functionally verified tagged
protein. Complexes will be separated on coomassie 1D gels and identified by collecting LC-MS/MS data
on the peptide mixtures generated by proteolytic digestion of the samples using the cross-charged
service offered by the Mass Spectrometry Laboratory. Samples will be purified from at least two distinct
breast tumour lines, running three replicates (and controls). MageA2-interacting proteins common to
both lines will be prioritised for functional validation using further CoIP and siRNA silencing assays.
4. If MageB or C proteins can also contribute to tamoxifen resistance in the functional studies above,
then the associations between Mage expression and TR in tumour samples we have calculated to date
may be an underestimate. Therefore the tumour cohorts we have will be reassessed for expression of
additional Mage (B and C) proteins. We have additional cases of patients who relapsed on tamoxifen
treatment from Valerie Spears in Leeds which will also be assessed for Mage expression.
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Institute of Cancer School PhD Project Proposal
A short (200 word) summary of the project to go with the studentship advert.
The majority of breast cancer patients can be treated with tamoxifen, however, a significant proportion
will relapse with resistant disease. Newer endocrine therapies have been developed, but current clinical
data suggests that patients respond better if they receive tamoxifen for 2-3 years before moving to
another treatment. Therefore, research into the mechanisms behind tamoxifen resistance (TR) remains
highly relevant. We have observed upregulation of the cancer/testis antigen MAGEA2 in TR cell lines.
Expression of MAGEA2 in sensitive cells allowed continued proliferation in tamoxifen media, while
silencing MAGEA2 expression in TR lines restored sensitivity. Furthermore, MageA2 formed complexes
with p53 and inhibited its activation in tamoxifen media. Analysis of MageA expression in clinical samples
showed a correlation with poorer outcome and MAGEA2-expressing xenografts also grew in the presence
of tamoxifen confirming a functional role for MAGEA2 in resistance. The project will use a variety of
molecular biology and proteomic techniques to test the wider significance of these data by determining if
other MAGE genes can confer resistance. The cellular pathways acted on by Mage proteins will be further
explored using co-immunoprecipitation assays and testing for interaction with candidate genes from
known TR pathways, including ER itself and to use mass spectromotery to identify novel interactors. The
in vitro findings will be validated by reference to clinical samples.
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