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Project title:
Role of vimentin in epithelial-mesenchymal transition (EMT) during pathogenesis
of solid tumours.
Supervisor: Dr Ahmad Waseem (a.waseem@qmul.ac.uk)
Co-Supervisor:
Dr AnandLalli (a.lalli@qmul.ac.uk)
Background:
Epithelial mesenchymal transition (EMT) is a process in which epithelial cells
lose their characteristic features such as cell-cell adhesion, polarity and they
reorganise their cytoskeleton whereby synthesis of keratin proteins is reduced
and vimentin expression is increased. Cells undergoing EMT are migratory and
express matrix metalloproteinases, which gives invasive characteristics allowing
them to cross the basement membrane, and they become resistant to anoikis.
Following EMT cells differentiate into different cell types and in some cases back
into epithelial cells through a process know as mesenchymal-epithelial transition
(MET). EMT has been shown to play a key role in early embryogenesis
especially during gastrulation and neural crest migration. On the other hand MET
plays a vital role during organogenesis such as nephrogenesis, cardiogenesis,
hepatogenesis and somitogenesis.
Development of solid tumours begins with a single cell, which accumulates
genetic lesions, and starts to proliferate to form a mass of cells. In many respects,
a developing tumour resembles an early embryo, especially expression of genes
associated with early embryogenesis such as keratins K8, K18, FOXM1 and
genes for homeobox proteins. For a cancer to invade the surrounding tissues,
the tumour cells must select for signaling pathways that not only support cancer
cell survival but also support their migration, invasion and proliferation after
release from the primary tumour site. Normally, the detached epithelial
carcinoma cells entering into the microvasculature would be lost by shearing
forces in narrow capillaries. However, metastatic tumor cells are able to persist
through adapted signaling pathways and can tolerate the morphologic alterations
in cell shape, plasticity, and deformation that are necessary to escape and readhere at distant sites
In cancers at a more advanced stage the selected signaling pathways bring
about EMT, which is associated with tumour invasiveness or metastasis. The
EMT during tumour invasion is remarkably similar to that during embryogenesis
in which the epithelial phenotype of tight intercellular junctions and polarity
across the epithelial layer is replaced by a mesenchymal phenotype with reduced
cell-cell adhesions, altered shape, expression of mesenchymal markers, and
enhanced cell motility. Thus, EMT is a hallmark of tumor progression,
characterizing highly invasive and metastatic carcinomas. A number of key
transcription factors are associated with the EMT process, including Snail, Slug
and Twist, as well as repressors of E-cadherin, ZEB1 and ZEB2.
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The most significant biomarker of mesenchemal tissue is the expression of
vimentin, a type III intermediate filament protein, which forms homopolymeric
cytoplasmic filaments. Several studies have shown that vimentin expression is
increased in cancer tissues undergoing EMT whereas keratin expression is
reduced. For example, squamous carcinoma cells expressing low levels of
keratin K19 are more invasive, but this is decreased by ectopic K19 expression.
In addition, K14 downregulation has been documented in a cervical cancer. We
have shown that vimentin expression during EMT is not a mere biomarker
instead it is responsible for reducing keratin expression. Using siRNA to inhibit
vimentin expression in invasive and tumorigenic head and neck squamous
carcinoma (HNSCC) cells, we have shown inhibition of cell growth and motility in
vitro, as well as in vivo tumour growth in athymic mice. Furthermore, we found
that small tumours developing from vimentin-knockdown cells showed features of
epithelial re-differentiation. This was further supported by our observation that
keratin promoter activity was increased in vimentin-knockdown cells.
Although the underlying mechanism by which vimentin suppression inhibits
tumour growth and enhances MET is not known, it certainly has potential for a
future therapeutic approach. A detailed understanding of the mechanisms
regulating EMT (and MET) will identify targets for therapeutic intervention and
help to control aggressive malignant cancers.
Aims of the study:
This project is based on the hypothesis that vimentin expression plays an active
role in inducing EMT during oncogenesis. We have shown that vimentin
suppresses keratin expression proteins and drives metastasis. The specific aims
of the project are:
1. To test whether vimentin filament formation in the cytoplasm is the trigger
for EMT.
2. To identify the vimentin domain(s) necessary to suppress keratin
expression and induce EMT.
3. To Test whether vimentin expression induces invasiveness in
keratinocytes using organotypical cultures.
4. To identify genes those are influenced by vimentin expression in epithelial
cells.
Research training to be provided during the project:
The student working on this project will be registered for PhD with the Queen
Mary Doctoral College with two supervisors, a primary supervisor, Dr Ahmad
Waseem, and a co-supervisor, Dr Anand Lalli. The role of the co-supervisor will
be to provide research supervision in the absence of the primary supervisor. The
PhD student working on this project will receive two main forms of research
training:
Training specific to the project: The candidate will be undertaking the
most modern cell and molecular biology techniques such as cloning and
expression of eukaryotic genes, retroviral induced gene transfer, quantitative cell
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migration and proliferation, gene expression methods including absolute
quantitative PCR, western blotting, ELISA and organotypic cultures. Genome
wide gene expression and associated statistical analysis will be carried out at the
Genome Centre based at Barts campus. The supervisors will provide this form of
research training.
Training in research skills: In addition, the student will also receive training
in transferable skills essential to becoming a competent researcher. This type of
training will be provided by a group of lecturers as a core course to the postgraduate research programme, compulsory to all postgraduate students. This
will include an introductory course covering the role of supervisors, moral and
ethical issues, the law and basic skills (experimental design and scientific
presentation etc). This will be followed by a series of short courses on animals in
research, health and safety issues and a molecular biology workshop. As part
of the core training, the student will attend a detailed course on communication
skills including IT, a short course on statistics and practical training in writing a
thesis and grant proposals, as well as making formal presentations.
PhD students are also required to present posters and give oral presentations at
the Institute level (PhD day and William Harvey day) in the first year, and at
national and international meetings in later years of their research training.
Research infrastructure: The PhD student will be based in the Blizard institute,
a state of the art research facility located at the Royal London Hospital campus,
Whitechapel (London). The institute is exceptionally well equipped for cell and
molecular biology research including cloning, mRNA and protein expression and
tissue culture. There are also core facilities run by specialists, but available to all
researchers, such as high-speed centrifugation including two ultracentrifuges,
imaging services and FACS facilities. The research laboratories are run by six
laboratory managers who deal with issues such as freezer breakdown,
equipment repair and calibration, health and safety, radiation protection,
consumable supplies etc. Genomics and proteomics facilities are also available
within Queen Mary at the Barts campus. All the core facilities are available to
researchers on pay as you go basis, which makes the services sustainable for
our institute.
Research environment: The primary supervisor of this project is based in the
Blizard Institute, which has a unique design where there are no barriers between
disciplines, as well as basic scientists and clinicians, which creates an excellent
environment for PhD students to learn and grow from others experiences. The
Cancer Research PhD group meets on a regular basis for Journal Club that gives
all an opportunity to discuss their research as well as laboratory issues.
Our Institute has an excellent programme of encouraging PhD students to realise
their full potential. The Institute organizes the annual PhD day, which gives an
opportunity to all PhD students registered within the Institute to present their work
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either orally or through a poster. To incentivise the PhD programme, the Institute
gives prizes for the best presentation and the best poster.
Supervisory team: Dr Waseem has an excellent track record of supervising
PhD students; all past 7 PhD students have completed their PhDs in FOUR
years and have published papers in high impact journals. Dr Lalli is a newly
appointed Clinical Academic Lecturer and this will be his 1st PhD supervision.