Structured Report for the Pathological Society Awards: Small Grant Award
Grant Reference No: SGS 2010/05
Title: ‘The role of brachyury in the development and progression of chordoma’
Name and Address: Dr Nischalan Pillay, UCL Cancer Institute, 72 Huntley Street, London,WC1E
6BT
Background:
Brachyury, a transcription factor, has been implicated in the pathogenesis of familial chordoma and
more recently in a subset of sporadic chordomas as well. Despite this, little is known about how this
evolutionary conserved transcription factor mediates its effect in chordomas.
Currently the mainstay of treatment which are surgery and proton therapy for sacral and clival-based
chordomas respectively, has reached a plateau. Systemic therapy is largely ineffective. Patients
afflicted with this malignancy are prone to multiple recurrences, significant morbidity with eventual
metastasis and mortality. Particularly as this is an “orphan” cancer, there is an urgent need to identify
new targeted therapeutic interventions as there will never be sufficient number of patients for a large
randomised trial.
To date, upstream regulators and downstream targets of brachyury in mammalian notochord have not
been identified. Targets of brachyury in chordoma are also unknown and may potentially be
“druggable” hence providing a new avenue for therapy for this disease. They may also help to
understand the mechanisms by which brachyury effects its actions.
Original aim
To identify downstream targets of brachyury in chordoma.
Amendments
Soon after the proposal for the grant application was drawn up, a timely research paper implicating
P63 in the regulation of brachyury in murine cell lines and osteosarcomas was published(1). In view of
the topical nature of the subject and our interest in brachyury regulation we also sought to investigate
the role of P63 in human chordomas and osteosarcomas.
Progress report
1. Upstream regulators of brachyury
Title: P63 Does Not Regulate Brachyury in Chordomas and Osteosarcomas
Presented as a poster at Pathological Society Winter Meeting in Hinxton, Cambridge
(6,7thJanuary 2011).
Manuscript in press (Histopathology)
The aim of this study was to investigate whether expression of P63 accounts for the aberrant
expression of brachyury in human chordomas and to determine if brachyury was expressed in
osteosarcomas.
We screened 26 chordomas and the UCH1 human chordoma cell line for P63 mRNA expression by
RT-PCR initially using a PAN P63 primer set, and thereafter algorithmically using sets of transcript
specific primers for the TAP63 and deltaNP63 transcripts. All 27 cases showed expression of pan P63
but were negative for the deltaNP63 transcript. Immunohistochemistry, using the panP63
antibody4A4, was performed on a tissue microarray containing 50 chordomas including the 26 cases
analysed by RT-PCR. None of the cases showed P63 expression. In addition we screened a tissue
microarray, containing 200 osteosarcomas, for the expression of both brachyury and deltaNP63.
None of the cases showed immunoreactivity for these antibodies.
While it is intriguing that P63 is involved in the transcriptional regulation of brachyury in murine
embryos we have excluded this molecule as a transcriptional regulator of brachyury in chordomas, all
of which express this protein. Furthermore, neither brachyury nor P63 are expressed in human
osteosarcomas. In summary, the finding that the deltaNP63 isoform in mice regulate the expression of
brachyury cannot be translated to humans in the context of chordoma and osteosarcoma
development.
2. Downstream targets of brachyury
Comprehensive genome-wide transcription factor binding maps are rapidly being constructed and are
providing new insights into transcriptional regulation. One of the methodologies increasingly being
employed is chromatin immunoprecipitation followed by sequencing (ChIP-seq) . However this does
not provide information regarding whether the binding of the transcription factor is functional. One of
the strategies used to extract this information is to alter the level of the transcription factor in question
in cells and examine its target gene expression profile against the binding site information.
Given that the human chordoma cell line U-CH1 expresses brachyury at both the mRNA and protein
levels and that it is the only well characterized chordoma cell line that is currently available, we opted
to use it as for our experimental model. In order to identify direct transcriptional targets of brachyury in
this cell line, chromatin immunoprecipitation utilizing a specific antibody followed by next generation
sequencing was employed to identify Brachyury bound DNA. To select potentially functional direct
targets, this gene list was overlapped with the gene expression list generated from the brachyury
mRNA knockdown cells. Thus, utilizing prior biological knowledge and bioinformatics we have been
able to identify a consensus motif for brachyury binding in a human cell line, identify direct
transcriptional targets of brachyury and develop further hypothesis regarding its role in the biology of
the disease.
Using this approach we have catalogued for the first time the genome-wide binding events for
brachyury in a mammalian cell line, identified potential “druggable” targets and gained significant
insight into the role of brachyury in the pathogenesis of chordoma. Given the emerging role of
brachyury in other cancers for example colorectal cancer, this work may serve as a resource for
others researching brachyury’s oncogenic properties.
How closely have the original aims been met:
The generous grant from the Pathological Society has enabled us to meet our original aim of
identifying downstream targets of brachyury but additionally we were able to investigate a potential
upstream regulator as well. The work on the targets is being prepared for publication in a high impact
peer review journal in which the funding from the Pathological Society will be acknowledged.
I would like to thank the Pathological Society for their support.
Nischalan Pillay
Reference:
1. Cho MS, Chan IL, Flores ER. ΔNp63 transcriptionally regulates brachyury, a gene with
diverse roles in limb development,tumorigenesis and metastasis. Cell Cycle 2010;9:1-8.