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Reguation of cancer stem cell stemness and senescence by homeobox
protein HOXA7.
Supervisor: Dr Ahmad Waseem (a.waseem@qmul.ac.uk)
Co-Supervisor: Dr Anand Lalli (a.lalli@qmul.ac.uk)
A) The clinical problem: Head and Neck (HNSCC) cancer is the sixth most common cancer in the World
with more than half a million new cases diagnosed each year (Ferlay et al., 2010). Survival rates are poor
(10-30%) among patients presenting with advanced disease (Haddad and Shin, 2008) and the majority of
HNSCC are diagnosed at a late stage. Despite the incidence of other common cancers such as lung, skin
and breast coming down year on year in the United Kingdom, the number of HNSCC cases continues to rise
(OCIU 2010 - Profile of Head and Neck Cancers in England: Incidence, Mortality and Survival). Worldwide
incidence is also rising and likely to reach to 750,000/year by 2015 (Ferlay et al., 2010). The most worrying
aspect is that more and more younger age groups are being affected by HNSCC (Shiboski et al., 2005).
HNSCC treatment modalities involve a combination of radical surgical intervention, high-dose radiotherapy
and systemic chemotherapy all of which cause significant morbidity for the 50% of patients who survive the
cancer at 5 years (OCIU 2010 - Profile of Head and Neck Cancers in England: Incidence, Mortality and
Survival). Survivors are often left with life changing side-effects of the cancer treatment such as disfiguring
facial scarring and the consequence of reduced salivation from radiotherapy related destruction of the
salivary glands such as difficulty in eating or increased incidence of dental pain.
On the other hand if cancer is diagnosed early then there is good chance the cancer could be removed
completely and patients could have a longer disease free life. Early diagnosis of HNSCC is extremely
difficult because there are several harmless lesions which look more or less similar to a pre-cancer oral
lesion. In 2013 we developed the quantitative Malignancy Index Diagnostic System (qMIDS) which involved
determining the cumulative expression of 16 genes for estimating the quantity of cancer cells in a HNSCC
sample (Teh et al., 2013). Most of these genes are associated with malignant transformation and therefore
understanding their functions will provide wealth of information about how cancer develops and spreads in
the body.
One of the key genes of the qMIDS is HOXA7, a member of the homeobox family of proteins, which is
involved in embryogenesis. This gene is expressed at very low levels in normal adult tissue but in precancer lesions its expression is induced to very high levels. Expression of HOXA7 is associated with
induction of growth, suppression of differention and senescence, the key features associated with
oncogenesis. In this study we propose to study the role of HOXA7 in development of cancer using a variety
of cell and molecular biology approaches.
B) Background about HOXA7 in cancer: HOXA7 is a member of the “A cluster” of homeobox genes
located on chromosome 7p15.2 and is highly homologous to the Antp gene in Drosophila. The protein has
230 amino acids with a molecular weight of approximately 25 kDa. HOXA7 is a transcription factor, which is
involved in gene expression, cell proliferation and differentiation. The protein contains a highly conserved
60-amino acid homeodomain with a helix-turn-helix motif, which is vital for DNA binding. HOX genes play a
vital role during embryonic development but their role in adults is not very clear. HOXA7 has been shown to
be associated with a number of cancers including acute leukemia, acute promyelocytic lekemia, breast
cancer and ovarian cancer.
In vitro studies have shown that downregulation of HOXA7 in MCF7, a breast cancer cell line, leads to
reduced cell proliferation and lower expression of the estrogen receptor ERα, whereas the overexpression
stimulated the estradiol (E2) related proliferation of cancer cells (Zhang et al., 2013). In ovarian cancer,
HOXA7 does not appear to induce differentiation of ovarian cancer cells directly but it rather promotes
differentiation via HOXA9, HOXA10, and HOXA11 (Cheng et al., 2005). However, in skin keratinocytes
ectopic expression of HOXA7 can repress 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced
differentiation by downregulating keratinocyte-specific differentiation gene, TGM1 (transglutaminase type 1).
Consistently, the TPA-induced differentiation suppressed expression of HOXA7 and its knockdown reversed
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the effects and resulted in elevated levels of differentiation markers (La Celle and Polakowska, 2001).
HOXA7 also induces growth in skin keratinocytes which together with suppression of differentiation
highlights its oncogenic role (La Celle and Polakowska, 2001). While the dysregulation of HOX plays a
major role in the pathophysiology of cancer, it has also been shown that not only the expression of HOX
genes but also their localisation is involved in cancer pathology. HOX proteins can be located in both nuclei
and cytoplasm (Corsetti et al., 1995; Kosaka et al., 2004; Naora, 2005; Naora et al., 2001; Ota et al., 2006).
Immunohistochemical staining shows HOXA7 presence in cytoplasm in cancer tissues, however,
approximately 78% of the cases showed nuclear staining, which correlated well with better prognosis. In a
tissue culture model the localisation of HOX protein can be influenced by various factors, e.g. truncated
HOXB6 is found in the cytoplasm in undifferentiated keratinocytes, whereas the full-length HOXB6 is
localized in the nuclei (Komuves et al., 2000). The localisation can also be regulated by external stimuli, e.g.
HOXA9 shows nuclear translocation after induction with thrombopoietin (Kirito et al., 2004).
Microarray analysis has shown that HOXA7 is consistently induced in ovarian tumours. There was no
expression of HOXA7 in pre-cancer lesion but appeared in metaplastic and malignant tissues (Ota et al.,
2007). HOX proteins are also shown to be involved in mesenchymal-epithelial transition (MET) (Cheng et al.,
2005). The overexpression of HOXA10 in endometrial cancer cells reduced cancer invasion and tumour
dissemination by inducing the expression of E-cadherin as well as the down-regulation of Snail, which is a
repressor of E-cadherin (Yoshida et al., 2006). Similar effects of MET is seen in ovarian surface epithelial
cells as the overexpression of HOXA7 results in the downregulation of vimentin and upregulation of Ecadherin (Naora et al., 2001). MET is the reversal of epithelial-mesenchymal transition (EMT), one of the
hallmarks of cancer metastasis (Hanahan and Weinberg, 2011). EMT allow loss of tight organization of
epithelial cells which enables them to invade surrounding tissue as well as into the adjacent blood vessels
or lymphatic channels in cancer patients (Hanahan and Weinberg, 2011).
C) Research work that led to the project: In a previous study we have developed a method in which the
cumulative expression of 16 genes is converted into an index called Quantitative Malignancy Index System
or qMIDS, which can differentiate between normal, dysplastic and malignant tissue (Teh et al., 2013). The
index values of 2, 4 and 8 were for normal, dysplatic and malignant tissues, respectively. The higher the
index, the higher the percentage of cancer cells in a tumour sample.
One of the key genes that were used to calculate qMIDS, is HOXA7. This gene plays a key role in
embryonic development but in adults HOXA7 is not expressed (Ota et al., 2007). Ectopic expression of
HOXA7 fused with Green Fluorescent Protein from Aequorea coerulescens (AcGFP) in a retroviral vector
pLPC-N_AcGFP showed suppression of differentiation, increase in stemness without significant effect on
clonogenicity.
The evidence for a role of HOXA7 in senescence comes from the observation that h-TERT is a downstream
target for HOXA7 in various forms of leukaemias (Gessner et al., 2010). The h-TERT promoter has a
specific binding site for HOXA7 and the interaction of HOXA7 with h-TERT promoter induces h-TERT
expression and suppresses senescence. Whether a similar mechanism operates in the development of
HNSCCs remains to be established.
D) Hypothesis to be tested: The purpose of this project is to test our hypothesis that HOXA7 induces
oncogenesis by increasing keratinocyte stemness and at the same time suppressing their ability to undergo
senescence. The aims of the project are as follows:
1. To develop an inducible system for HOXA7 ectopic expression for functional analysis.
2. To determine the influence of HOXA7 on the growth and differentiation of keratinocytes in an
organotypic culture model.
3. To determine the global changes in keratinocyte gene expression induced by HOXA7 ectopic
expression.
4. To determine whether ectopic expression of HOXA7 in keratinocytes increases telomere length
and telomerase activity.
5. To determine the role of HOXA7 in inducing MET in cancer cells.
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E) Research training to be provided during the project: The student working on this project will be
registered for PhD with the QM Doctoral College with two supervisors, a primary supervisor (the applicant)
and a co-supervisor. 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 types 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 migration and proliferation, gene expression methods including absolute
quantitative PCR, western blotting, ELISA, telomerase activity assays 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 type 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 post-graduate 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.
F) Research infrastructure: The PhD student will be based in the Blizard institute, state of the art research
facility located at the Royal London Hospital campus, Whitechapel. The institute is exceptionally 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.
G) Research environment: The primary supervisor of this project is employed by the Institute of Dentistry
but his research laboratory is based in the Blizard Institute. The Institute has a unique design where there
are no barriers between disciplines, which creates an excellent environment for PhD students to learn and
grow from others experiences. The Institute of Dentistry has an excellent programme of encouraging PhD
students to realize 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 either orally or through a
poster. To incentivise the PhD programme, the Institute gives prizes for the best presentation and the best
porter on the PhD day. The PhD day is led by the Director of Graduate Studies in Dentistry and attended by
all staff including the Dean of the Dental Institute.
H) Public engagement about our research?: In line with the Queen Mary University of London policy to
inspire curiosity and learning by conveying science to the public, we engage the public with our research
findings through “The Centre of the Cell” initiative (www.centreofthecell.org; Director, Professor Frances
Balkwill) which was set up in 2006 by the School of Medicine and Dentistry, solely for this purpose. The
Blizard Institute building, where our laboratory is situated, is the only public exhibition space in the world
located within a research building. Through this unique interactive multimedia experience, embedded within
a research environment where we are based, our research has an impact on educational and career
aspirations of many young people. In addition to conventional refereed research publications, we also
disseminate our research outcome to the public, by publishing our research outcome in the Institute’s
Internet website to provide current research activities and explain in layman terms how our research is likely
to benefit patient care.
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The College has a press office to deal with significant findings that can be communicated to the public
through press releases.
I) Record of PhD supervision by the applicant: The primary supervisor has an excellent track record of
supervising PhD students; all past 7 full-time PhD students 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. The supervision style is mostly informal with one to one regular meetings so
any problem faced by a student is resolved promptly.
J) References:
Cheng W, Liu J, Yoshida H, Rosen D, Naora H (2005) Lineage infidelity of epithelial ovarian cancers is
controlled by HOX genes that specify regional identity in the reproductive tract. Nat Med 11:531-7.
Corsetti MT, Levi G, Lancia F, Sanseverino L, Ferrini S, Boncinelli E, et al. (1995) Nucleolar localisation of
three Hox homeoproteins. J Cell Sci 108 ( Pt 1):187-93.
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM (2010) Estimates of worldwide burden of
cancer in 2008: GLOBOCAN 2008. Int J Cancer 127:2893-917.
Gessner A, Thomas M, Castro PG, Buchler L, Scholz A, Brummendorf TH, et al. (2010) Leukemic fusion
genes MLL/AF4 and AML1/MTG8 support leukemic self-renewal by controlling expression of the
telomerase subunit TERT. Leukemia 24:1751-9.
Haddad RI, Shin DM (2008) Recent advances in head and neck cancer. N Engl J Med 359:1143-54.
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646-74.
Kirito K, Fox N, Kaushansky K (2004) Thrombopoietin induces HOXA9 nuclear transport in immature
hematopoietic cells: potential mechanism by which the hormone favorably affects hematopoietic
stem cells. Mol Cell Biol 24:6751-62.
Komuves LG, Shen WF, Kwong A, Stelnicki E, Rozenfeld S, Oda Y, et al. (2000) Changes in HOXB6
homeodomain protein structure and localization during human epidermal development and
differentiation. Dev Dyn 218:636-47.
Kosaka Y, Akimoto Y, Yokozawa K, Obinata A, Hirano H (2004) Localization of HB9 homeodomain protein
and characterization of its nuclear localization signal during chick embryonic skin development.
Histochem Cell Biol 122:237-47.
La Celle PT, Polakowska RR (2001) Human homeobox HOXA7 regulates keratinocyte transglutaminase
type 1 and inhibits differentiation. The Journal of biological chemistry 276:32844-53.
Naora H (2005) Developmental patterning in the wrong context: the paradox of epithelial ovarian cancers.
Cell Cycle 4:1033-5.
Naora H, Montz FJ, Chai CY, Roden RB (2001) Aberrant expression of homeobox gene HOXA7 is
associated with mullerian-like differentiation of epithelial ovarian tumors and the generation of a
specific autologous antibody response. Proc Natl Acad Sci U S A 98:15209-14.
Ota T, Choi KB, Gilks CB, Leung PC, Auersperg N (2006) Cell type- and stage-specific changes in HOXA7
protein expression in human ovarian folliculogenesis: possible role of GDF-9. Differentiation 74:1-10.
Ota T, Gilks CB, Longacre T, Leung PC, Auersperg N (2007) HOXA7 in epithelial ovarian cancer:
interrelationships between differentiation and clinical features. Reproductive sciences 14:605-14.
Shiboski CH, Schmidt BL, Jordan RC (2005) Tongue and tonsil carcinoma: increasing trends in the U.S.
population ages 20-44 years. Cancer 103:1843-9.
Teh MT, Hutchison IL, Costea DE, Neppelberg E, Liavaag PG, Purdie K, et al. (2013) Exploiting FOXM1orchestrated molecular network for early squamous cell carcinoma diagnosis and prognosis.
International journal of cancer Journal international du cancer 132:2095-106.
Yoshida H, Broaddus R, Cheng W, Xie S, Naora H (2006) Deregulation of the HOXA10 homeobox gene in
endometrial carcinoma: role in epithelial-mesenchymal transition. Cancer Res 66:889-97.
Zhang Y, Cheng JC, Huang HF, Leung PC (2013) Homeobox A7 stimulates breast cancer cell proliferation
by up-regulating estrogen receptor-alpha. Biochemical and biophysical research communications
440:652-7.