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2009 Imperial College Junior Research Fellowship Application Section A

(To be completed by Applicant in Arial pt 11)

Project Title Study of OPCML tumour suppressor functions in epithelial ovarian cancer in the context of FGF signalling. Fellowship Applicant Details Name Evangelos Ntougkos Title/Position Dr/Postdoctoral research associate University/Institution Hellenic Pasteur Institute Address (for correspondence)

1 Platia Kypselis Athens 11361 Greece

Email vangelis_nt@yahoo.com Telephone +306945786560 I certify that the statements herein are true, complete and accurate to the best of my knowledge. I agree to accept responsibility for the scientific content of the project and to provide the required progress reports if a fellowship is awarded as a result of this application Signature of Applicant: Date: Sponsor Details Name Hani Gabra Title/Position Professor of Medical Oncology/Director of the Ovarian Cancer

Action Research Centre Faculty Medicine Email h.gabra@imperial.ac.uk I agree to provide relevant equipment and laboratory space for the Applicant’s research proposal and ensure that there is no additional cost burden on the College. I also agree to ensure that before any research funded by the Fellowship commences and during the full award period, all the necessary legal and regulatory requirements in order to conduct the research are met, and all the necessary licences and approvals have been obtained.

Signature of Sponsor: Date: Head of Department/Division (HoD) Details Name Charles Coombes Title/Position Professor/Head of Department of Oncology Faculty Medicine Email c.coombes@imperial.ac.uk I understand that this scheme will not be responsible for continued support past the three years and that any costs around the ending of employment that result (directly or indirectly) in consequence of the scheme's limited fixed funding will need to be borne by the employing department/division. Signature of Head of Department/Division: Date:

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Proposed Start Date (projects must start within 3 months of October 2009) November 2009 Ethics Will animals be used? No Are licences already in place? Yes

Will human subjects or samples be used? Yes Is regulatory approval already in place? Yes

Collaborators (not sponsors, list name(s) and attach letter(s) of support) Lay summary (maximum 300 words) Ovarian cancer is one of the commonest types of cancer in women. Although if diagnosed early patients can be given effective treatment options, it is more often detected at a later stage when curative treatment is less likely. Management involves surgery followed by chemotherapy but unfortunately many tumours after a while do not respond to chemotherapy. OPCML is a gene responsible for the production of a protein which we believe has a protective role against the development of various types of cancer, notably ovarian cancer. We know that healthy cells have this protein and hence benefit from its protective functions; however when ovarian cells produce very low levels of it a tumour may form. It is thought that by “switching off” this gene cells evade mechanisms which keep them normal and thus acquire certain characteristics that can eventually make them cancerous. Although we are beginning to know what OPCML does, we do not understand the mechanisms behind its protective roles. One of the ways that proteins work is by associating with other proteins either in a cooperative or disruptive way. There is evidence to suggest that OPCML potentially obstructs the functions of a group of proteins that are responsible for features that cells need to have in order to become cancerous. So, in circumstances when control proteins such as OPCML are lost, cells can acquire these abnormal features and development of a tumour can occur. The name of the group that we think is normally controlled by OPCML is the fibroblast growth factor proteins. The aim of this project is to study the way OPCML and the FGF proteins interact. The ultimate goal is that, having understood how OPCML normally controls the FGF proteins and what happens when this control is lost, we could offer better options for diagnosis, treatment prediction, prognostic testing and treatment in the future that would target FGF and the restoration of its control, either alone or alongside standard current therapies.

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Section B: Budget (To be completed by Applicant in Arial pt 11)

Current Salary in £ 17,000

Source of Current Salary Programme Grant (Greek-EU)

Please expand table as necessary

Category Breakdown by item headings Category Total Consumables (to include costs of animals where required)

1) General laboratory consumables 2) 3)

8,500 x 3

Travel 1) AACR Annual Meetings 2) 3)

1,500 x 3

Miscellaneous (to include equipment, access charges where required)

1) 2) 3)

Total 10,000 x 3

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Section C: Research Proposal (To be completed by Applicant in Arial pt 11)

Please complete each section and expand as necessary to a maximum of 5 pages not including references, graphs and figures. 1) Summary (Include the key question that you wish to answer, and scientific summary to include: background, hypothesis, and expected outcomes) Epithelial ovarian cancer (EOC) is a very common cause of death from gynaecological malignancy. It arises in the monolayer of cells overlying the ovary called the ovarian surface epithelium. Although if diagnosed early patients have a good prognosis, it is most often diagnosed at advanced stages of disseminated disease when the chances of survival are very low. Management usually consists of surgery followed by platinum-based chemotherapy. OPCML has been proposed as a novel tumour suppressor gene in sporadic epithelial ovarian cancer. OPCML is normally expressed in the ovarian surface epithelium, but is frequently epigenetically silenced in EOC by somatic CpG island methylation. Moreover, it exhibits functional features typical of a tumour suppressor: it suppresses cell growth in vitro and tumourigenicity in vivo. The underlying mechanism of OPCML’s tumour suppressor phenotype in EOC has been until recently evasive. Recent evidence suggests that one of the connections between OPCML and ovarian cancer lies in its involvement as a modulator of receptor tyrosine kinase (RTK) signalling including the FGF system. The FGF signalling system is made up by a group of RTKs and growth factor ligands, which impact on a multitude of physiological processes and pathologies. The proposed project is mainly an attempt to identify the way OPCML interacts with the FGF system and how this interaction is impaired in EOC. Questions will include the nature of this interaction, its effects on signalling pathways and how these signalling pathways can account for the phenotypes associated with OPCML (or its functional absence). A successful completion of the proposed project will offer a new level of understanding of the biology of OPCML, and its pathophysiology in EOC, a disease that at the molecular level is poorly understood. The value of expected outcomes, i.e. of a better understanding of normal regulation by OPCML of the FGF pathway in the ovary and the dysfunction of this system in cancer, can be appreciated thanks to its potential therapeutic translatability: targeting the interaction between OPCML and its FGF partner could offer a much awaited new treatment modality for patients of EOC. Furthermore, a better understanding could aid in the development of biomarkers that could be used for early diagnosis, therapeutic response prediction when targeting the FGF pathway and also prognostic prediction for individual patent management.

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2) Background (Provide context for the application, including research in the field and by the applicant and supervisors, explain why you are especially qualified to carry out the proposed research) Ovarian cancer is second most frequent cause of death from gynaecological malignancy globally [1]. It ranks fourth as a cause of death from cancer in women. The incidence of the disease rises with age, peaking in around the seventh to eight decade. 90% of ovarian cancers are epithelial ovarian cancers (EOCs), the rest originating in stromal and germ cells. Cancer of the ovary is primarily a sporadic disease, with familial cases accounting for only 5-10% of patients [2, 3]. EOC arises in the single-cell layer overlying the ovary termed the ovarian surface epithelium [4]. This inconspicuous cell layer developmentally is part of the coelomic epithelium, while in the adult it functions in transporting materials to and from the peritoneal cavity and playing an important role in cycles of ovulation and post-ovulatory repair. Ovarian tumours are among the most complex in terms of histogenesis and are classified into serous, endometrioid, mucinous, clear cell and other more uncommon types. There are four basic stages of advancement in ovarian cancer, stage I being limited to the ovary, stage II involving the pelvis, stage III the peritoneum, while stage IV being characterised by distant metastases. It should be noted that ovarian cancer on the whole is not overtly a visceral metastatic disease, but is commonly associated with locoregional peritoneal dissemination. Treatment of ovarian cancer varies according to the stage of its detection, but commonly involves surgical debulking and platinum-based chemotherapy [5]. Two major hypotheses have been proposed for the aetiology of ovarian cancer. The incessant ovulation hypothesis is based on the idea that continuous ovulatory cycles are damaging to the ovarian surface epithelium [6]. The pituitary gonadotrophin hypothesis posits that entrapped epithelium within the stroma is abnormally stimulated by factors such as oestrogen and oestrogen precursors in the presence of high and persistent levels of gonadotrophins favouring the formation of neoplasms [7]. Increasingly, inflammation has been regarded as an essential element in the aetiology of ovarian neoplasia, probably through the same processes that physiologically tie it to ovulation [8]. One of the genes that has been proposed to act as a tumour suppressor in sporadic EOC is OPCML (opioid binding protein / cell adhesion molecule-like) [9]. OPCML is part of the IgLON family of neural glycoproteins, which are cell adhesion molecules of the immunoglobulin superfamily [10]. This family, apart from OPCML, includes HNT (neurotrimin), LSAMP (limbic-system associated membrane protein) and NEGR1 (neuronal growth factor regulator 1). The OPCML protein (also known as OBCAM) was originally purified from rat brain as a μ opioid-specific receptor, but eventually it was realised that it did not fit the criteria of an opioid receptor [11, 12]. OPCML undergoes post-translational N-linked glycosylation and, being entirely extracellular, is tethered to the cell membrane by a GPI anchor. Moreover, the OPCML protein is detected in the Triton-insoluble fraction of cell membrane preparations, a fraction that is associated with lipid rafts [13]. OPCML exhibits limited expression in some organs in the adult, while it is more strongly expressed in the brain and the ovary; in the latter, mostly in the epithelial component [9]. In both the rat and the chick OPCML expression is known to rise during development [13, 14]. OPCML can bind homophilically but also heterophilically with other members of the IgLON family. Although originally thought to influence axon guidance by neurite growth, more recent evidence suggests it might be primarily involved in cell-cell adhesion and cell recognition [15]. OPCML has been implicated in visual cortex plasticity and indirectly in the inflammatory response [16, 17]. With regard to heterophilic interactions within the IgLON family, it has been proposed that each IgLON is principally a subunit of a heterodimeric adhesion molecule, termed Diglon [18]. The relationship between OPCML and ovarian cancer was identified by Gabra and colleagues who proposed it as a novel tumour suppressor gene in EOC [9]. OPCML was originally identified through a high rate of complete loss of heterozygosity within its second intron on 11q25. OPCML was shown to be expressed in the normal ovarian epithelium, the site of origin of EOC, but its RNA expression was abolished in

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the vast majority of both a panel of ovarian tumours and a panel of ovarian cancer cell lines. Extensive mutation screening identified only one somatic mutation in EOC, a C→G transversion that results in a missense P95R substitution in the first immunoglobulin domain of the protein. Nonetheless, very frequent promoter hypermethylation was found in ovarian tumour samples and cancer cell lines suggesting that the mechanism underpinning OPCML silencing is epigenetic. Furthermore, it was shown that OPCML has functional features of a tumour suppressor: it suppresses cell growth in vitro, when expressed in the ovarian cancer cell line SKOV-3, and tumourigenicity in vivo, in intraperitoneal and subcutaneous nude mouse xenografts. Since the first publication on the link between OPCML and ovarian cancer, there have been many other papers with data that associate OPCML with ovarian and other cancers, as well as other IgLONs. Ntougkos, Gabra and colleagues examined the expression of all four IgLONs in EOC and found that apart from OPCML, LSAMP and NEGR1 are also down-regulated in EOC, whereas the expression of HNT is higher than in normal samples [19]. Reduced OPCML expression and/or promoter hypermethylation has been demonstrated at high frequency in other human cancers, such as lung, nasopharyngeal, cervical and brain tumours [20-22]. Another IgLON, LSAMP was shown to be down-regulated in renal cell carcinoma cell lines and samples in comparison to the normal kidney, also due to promoter hypermethylation [23]. The fibroblast growth factor (FGF) superfamily comprises 23 genes in humans encoding structurally-related proteins with high affinity to heparin, including its prototype members FGF1 and FGF2 [24, 25]. This superfamily is involved ubiquitously in development and in diverse process in the adult, both physiologically and in pathological conditions [26, 27]. Most FGFs are mitogenic, can stimulate cell migration and differentiation and are considered to be survival factors [28, 29]. They are involved in wound healing, angiogenesis, tissue repair and other homeostatic processes. Their biological activity is exerted by binding to and activation of high-affinity cell-surface FGF receptors (FGFR1-4) that have intrinsic tyrosine kinase activity. Binding results in receptor dimerisation and kinase domain autophosphorylation [30]. In terms of signalling, the FGF system broadly encompasses MAPK pathways, PI3K pathways, PLCγ pathways and Src kinases, thus indicating the broad spectrum of processes in which it is involved. Most FGF superfamily members contain a heparin and heparin sulphate-binding domain, hence by binding to heparin sulphate proteoglycans (HSPGs) at the cell surface they are protected against proteases and thermal denaturation and their interaction with FGFRs is regulated [31, 32]. In addition, FGFRs signal through the FRS2 family of docking/scaffold adaptor proteins, a fact which adds to their level of regulation but also complexity [33]. The involvement of the FGF system in EOC has been implicated through an accumulated volume of evidence. The expression of FGF and FGFR members has been found to be increased often through genomic amplification [34-36]. FGF2 has been validated as a serum biomarker for EOC [37]. Although drug development based on the FGF pathway has been undertaken, no treatment targeting this pathway is as yet offered in the clinic. Since the initial work on OPCML and ovarian cancer, progress has been made in deciphering the basis of this relationship. Contribution to this progress has been made initially through the work of the candidate under the supervision of the sponsor and subsequently by the group of the sponsor. Two notable findings through unpublished work by the applicant are pertinent to this application. Firstly, it has been demonstrated that OPCML promotes cell-cell adhesion, probably through heterophilic interactions (Fig.1). Moreover, based on evidence with respect to proliferation and apoptosis, the suppression effect of OPCML on cell growth is hypothesised to be mediated by apoptosis, as indicated by increased steady-state levels of apoptosis and sensitisation to the topoisomerase I inhibitor camptothecin (Fig.2). In the above experiments, the presence of the clinically occurring

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P95R mutation did not impair the function of OPCML. The sponsor’s group has addressed whether there is a potential link between OPCML, this novel tumour suppressor in ovarian cancer, and the FGF system, a signalling network involved in this disease as mentioned above. An array of interesting pilot data has been gathered. Notably, OPCML expression was found to down-regulate the expression of FGF2 at the protein level, but not at the RNA level (Fig.3, 4). Interestingly, OPCML expression also changed the relative abundance of FGF2 isoform proteins. Moreover, FGF2 stimulation of SKOV-3 ovarian cancer cells induced expression of OPCML both at the RNA (Fig.5) and the protein (Fig.6) level; the latter is detected with a lag compared with the former indicating the time needed for the OPCML message to be transcribed. This indicates that the relationship between the tumour suppressor OPCML and the oncogenic FGF2 is bidirectional, making it likely to be part of a control loop that counterbalances growth signals from FGF2 with control of those signals by OPCML. Preliminary signalling experiments showed that OPCML can abrogate FGFR1 phosphorylation at key tyrosine residues following stimulation with FGF2 (Fig.7). Finally, recent work by Prof. Gabra’s group has demonstrated that OPCML can inhibit cell migration towards FGF1 (Fig.8). The above previous work by the candidate is part of the candidate’s considerable experience in the field of OPCML and ovarian cancer, a field which is currently not studied in the UK by any group other than the sponsor’s. Hence, the proposed project is in essence a development from the candidate’s PhD, which identified interesting phenotypic aspects of OPCML, in the light of pilot data acquired by the sponsor’s current group that now link OPCML with the FGF signalling system.

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FIGURES

Figure 1 Effect of OPCML in cell-cell adhesion Percentage cell-cell adhesion in SKOV-3 OPCML cell lines as quantified by adhesion assays. Cells under test were fluorescently-labelled and allowed to attach on unlabelled cell monolayers for 90 min. Values are corrected for background fluorescence. Error bars represent the SEM of three independent experiments. * p < 0.05, ** p < 0.01 compared to parental cell line by Student’s t test. WT = wild type OPCML transfected; Mut = mutant OPCML transfected; Par = parent.

Figure 2 Effect of OPCML on apoptosis Annexin V analysis in the SKOV-3 OPCML cell lines by flow cytometry, measuring steady-state levels of apoptosis (A), and after treatment with 1 μM camptothecin for 48 h (B). Error bars represent the SEM of three independent experiments; in each, a total of 30,000 events were acquired. * p < 0.05, ** p < 0.01 compared to parental cell line by Student’s t test. (Cell lines as in Fig.1)

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Figure 3 FGF2 mRNA expression in various cell lines FGF2 expression was examined at the RNA level by quantitative RT-PCR in two SKOV-3 clones transfected with OPCML (BKS2.1, SKOBS 3.5) and compared to the vector control (SKOBSV1.2) or the untransfected cell line; three non-ovarian cell lines were also examined: LNCaP, PC3 and DU145, all of prostate cancer origin.

Figure 4 FGF2 protein expression in SKOV-3 cells expressing OPCML FGF2 expression was examined at the protein level by western blotting in SKOV-3 cell lines (as in Fig.3). The three different molecular weights correspond to FGF2 isoforms. Beta actin was used as a loading control.

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Figure 5 OPCML RNA expression in response to FGF2 stimulation OPCML expression in SKOV-3 cells was measured by quantitative RT-PCR following stimulation with FGF2.

Figure 6 OPCML protein expression in response to FGF2 stimulation OPCML expression in SKOV-3 cells was detected by western blotting following stimulation with FGF2. Beta actin was used as a loading control.

Figure 7 FGFR1 phosphorylation status in relation to OPCML expression FGFR1 phosphorylation at different tyrosine residues was examined by western blotting in SKOV-3 cells transfected with OPCML (as in Fig.3) following stimulation with FGF2. Beta tubulin was used as a loading control.

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Figure 8 Effect of OPCML on chemotaxis to FGF1 Migration of SKOV-3 cells with or without OPCML expression (as in Fig.3) examined by chemotactic migration assays using FGF1 as a chemotactic factor. Error bars represent the SEM of two independent experiments.

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3) Aims of the project (Include specific hypotheses to be tested) The main aim of the proposed project is to understand the relationship between OPCML and the FGF signalling system in the context of epithelial ovarian cancer. Thus, the working hypothesis which forms the basis of this project is that OPCML interacts with a member of the FGF signalling network. The question that then emerges is what is its binding partner? Is OPCML connected to the FGF system directly or indirectly? We propose that the most likely link is a direct one, involving physical binding to an FGF receptor, and this is what we wish to explore. Apart from establishing this interaction, the other aspect of the proposed project involves the effects of this interaction on signalling. Any effects identified will be pursued in the context of their ability to explain what we know about OPCML phenotypes of proliferation, apoptosis, migration and adhesion. Furthermore, based on unpublished pilot data that demonstrate that OPCML can sensitise cells in response to drugs that inhibit receptor tyrosine kinase signalling in ovarian cancer, we postulate that the status of OPCML expression could be used as a predictive surrogate for the action of therapies that target transmembrane receptors, such as FGFRs. A secondary hypothesis is that OPCML expression in ovarian cancer is linked to that of FGF members and this will be explored at least with respect to certain of the FGF members. The role of other IgLONs, which are relevant in ovarian cancer, merits investigation in combination with the primary focus on OPCML. Finally, there are factors in the FGF system that add to its level of complexity, such as the above-mentioned HSPGs and FRS2 adaptor proteins, which without a doubt will play a role in signalling affected by OPCML. Exploration of such factors may provide valuable clues to the regulation of the OPCML-FGF interaction. 4) Experimental plan and methods (Details of routine methods are not required. If relevant, please specify numbers for experiments involving animals/humans etc) This project on the whole will be based on the use of transfected SKOV-3 cells expressing varying levels of OPCML, which were successfully used in the past by the applicant and are currently used by the sponsor’s group. In addition, we will use an immortalised normal ovarian surface epithelium cell line that expresses OPCML that can be considered as a closer “more normal” model to study ovarian cancer. Initially, all pilot data need to be reproduced in order to rule out any uncertainties and extend the scope of the experiments (for example, by including more time-points).The project we propose has three main arms: 1. Identification of the binding partner of OPCML in the FGF system Given our hypothesis that there is a direct link between OPCML and the FGF system, I will attempt to find the FGF receptor protein partner that OPCML can bind to. This will be done by co-immunoprecipitation experiments using antibodies against OPCML and various FGF receptors. Having established the binding partner, localisation and internalisation experiments will indicate the role of sorting in relation to function. Moreover, with the aid of OPCML/GST fusion proteins currently used by the sponsor’s group, in vitro interactions between OPCML and its binding partner can be studied in the presence or absence of drugs or antibodies that target the FGF system. The available constructs, which encode variable lengths of the extracellular portion of OPCML, will also point to the minimal domain structure necessary for these interactions. 2. OPCML modulation of FGF receptor signalling and functional significance There is preliminary evidence that OPCML abrogates FGF-induced activation of FGF receptors so it is important to identify which pathways are affected and how. The approach to be used involves an array of western blotting experiments under stimulation with various FGF factors and examination of the phosphorylation status of upstream (FGF receptor) and downstream signalling proteins (MAPK, PI3K etc) in the context of OPCML expressing and OPCML silenced ovarian cancer cells and normal ovarian surface epithelial cells. This wide

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approach will lead to a more focused examination of signalling changes in the pathways identified. Once we have identified these changes, we will attempt to examine if they can account for phenotypes associated with OPCML. For example, potential links with adhesion could involve the PLCγ arm of FGF signalling, whereas with apoptosis the PI3K arm. With respect to chemotactic migration, through migration assays and examination of chemotaxis-relevant receptors, we propose to explore the basis of the role of OPCML in obstructing migration. As regards cell-cell adhesion, fluorescence-based assays will be used to investigate how this aspect of cell behaviour is modulated by FGF stimulation in different OPCML expression backgrounds and how OPCML-FGF interactions in trans affect adhesion. The importance of the OPCML-FGF interaction will be explored in the context of apoptosis through Annexin V staining and other systems of detection by studying the effects of FGF stimulation in combination with OPCML status or blocking of FGF pathways. Finally, looking at the reverse direction of what appears to be a potentially regulatory loop, the effect of FGF2 on OPCML expression may be operating at different levels (intracrine, paracrine, etc) and an attempt to identify the relevant mechanisms could provide useful hints. 3. Diagnostic, predictive, prognostic and therapeutic use of the OPCML- FGF interaction in cancer In order to address the question of whether OPCML can be used as a surrogate marker for prediction of FGF-targeting therapy outcome, its expression will be monitored in ovarian cancer samples in connection with that of FGF and FGFR members. This will be undertaken through a quantitative RT-PCR study and immunohistochemistry using normal ovarian and ovarian cancer samples on tissue microarrays general at Imperial College. This approach will exploit OPCML as a surrogate diagnostically (in lieu of FGF2 for example) and in a predictive or prognostic manner in relation to FGF-based patient response and therapy outcome. In addition, the potential ability of OPCML to sensitise cells to FGFR inhibitors such as AZD2171 [38] will be examined by measuring cell death and abrogation of FGFR signalling. Two other general aspects will be considered in all the above investigations: the functional significance of the clinically occurring somatic P95R mutation, which can be explored by using the SKOV-3 cell line expressing this mutant form of OPCML, and the role of other IgLONs, especially LSAMP and NEGR1, in modulating the above signalling and phenotypic observations. 5) Justification for requested support (Reasons for the budget requested and include details of how funds will be obtained to support the research costs not covered by the College if necessary) The requested support is going to be mainly used to obtain the consumables necessary to carry out the proposed experiments. A small portion of the budget will be dedicated to travel expenses required to participate in national and international conferences in the field of cancer biology. 6) Where appropriate, describe potential project outputs that will lead to commercially exploitable results The main commercially exploitable outcome that could potentially stem from the proposed project is the use of OPCML as a predictive surrogate for treatments targeting FGFR in ovarian cancer. On a longer term basis, if an interaction between OPCML and an FGFR member is established, it could offer the option to develop therapeutic agents that can mimic this interaction and be therefore used as treatment modalities in the clinic.

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7) Describe how the award of an Imperial College Junior Research Fellowship would further your career? (up to 400 words)

The award of an Imperial College Junior Research Fellowship will benefit my career in many ways. It is without a doubt a prestigious position which would be a very important asset for an early-stage researcher. By providing the financial support to carry out a project independently, without having to engage oneself with administrative or teaching duties, it offers a unique opportunity for a scientist to be established at the most difficult career stage. The fact that the Fellowship is based at Imperial College, one of the world’s leading research institutions, gives the valuable advantage of an environment that fosters scientists to flourish and build on their skills and abilities. Furthermore, Imperial College represents a unique scientific setting where experts who are involved in cutting edge research can interact with each other across disciplines, exchanging ideas and views, on a fertile ground for the conception of novel ideas. The infrastructure of the College, which is outstanding, provides the means to test these ideas and translate them into actions. Most importantly, the fellowship programme is one of the few European programmes that provide the opportunity to bridge the gap between the postdoctoral level and a lectureship, which is one of the hardest obstacles in the career of a researcher. In addition, it relies on the relationship between the sponsor and the fellow, a relationship which has been voluntarily agreed upon in the interest of the fellow’s career development conferring to the fellowship a career-driven attribute. Finally, for a scientist dedicated to a research career, which is an uncertain path paved with competitive obstacles, it is precisely this competitiveness that having been surpassed renders the achievement of a junior research fellowship a remarkable landmark and the starting point of a more focused, independent phase, which can hopefully lead to a group leadership in the future.

8) Define the scientific considerations behind your choice of Sponsor (up to 200 words) The relationship that ties Prof. Gabra and me is a very strong one since he was the supervisor of my PhD, hence the first and most important mentor in my scientific career. The research subject of my PhD thesis has been pursued and furthered in his current laboratory. The proposed project for this fellowship can be viewed as a development from my PhD line of work in the light of recent significant findings by Prof. Gabra’s group. Therefore, not only do we share a common past, but more importantly we have the same interests and aspirations in research that aims at providing a sound understanding of ovarian cancer. In addition, Prof. Gabra has a unique profile of talents and achievements: he is a well-respected clinician in the field of gynaecological oncology, with significant accomplishments in the clinic, but also a world-renowned research leader in the area of ovarian cancer. The magnitude and value of the scientific environment that he is currently directing and building up for the future is unparalleled in the UK in the fight against ovarian cancer. As this is exactly what I wish to dedicate myself to in the future, there could be no better place for me to do this than with him.

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9) Publications cited (Provide full references including the titles, list alphabetically, and cite in the text by name or number, where possible include web-links) 1. Shibuya, K., et al., Global and regional estimates of cancer mortality and incidence

by site: II. Results for the global burden of disease 2000. BMC Cancer, 2002. 2: p. 37.

2. Stratton, J.F., et al., Contribution of BRCA1 mutations to ovarian cancer. N Engl J Med, 1997. 336(16): p. 1125-30.

3. Wenham, R.M., J.M. Lancaster, and A. Berchuck, Molecular aspects of ovarian cancer. Best Pract Res Clin Obstet Gynaecol, 2002. 16(4): p. 483-97.

4. Choi, K.C. and N. Auersperg, The ovarian surface epithelium: simple source of a complex disease. Minerva Ginecol, 2003. 55(4): p. 297-314.

5. Ozols, R.F., Treatment goals in ovarian cancer. Int J Gynecol Cancer, 2005. 15 Suppl 1: p. 3-11.

6. Fathalla, M.F., Incessant ovulation--a factor in ovarian neoplasia? Lancet, 1971. 2(7716): p. 163.

7. Cramer, D.W. and W.R. Welch, Determinants of ovarian cancer risk. II. Inferences regarding pathogenesis. J Natl Cancer Inst, 1983. 71(4): p. 717-21.

8. Ness, R.B. and C. Cottreau, Possible role of ovarian epithelial inflammation in ovarian cancer. J Natl Cancer Inst, 1999. 91(17): p. 1459-67.

9. Sellar, G.C., et al., OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer. Nat Genet, 2003. 34(3): p. 337-43.

10. Pimenta, A.F., et al., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting. Neuron, 1995. 15(2): p. 287-97.

11. Cho, T.M., et al., Purification to apparent homogeneity of a mu-type opioid receptor from rat brain. Proc Natl Acad Sci U S A, 1986. 83(12): p. 4138-42.

12. Hachisuka, A., et al., Characterization and tissue distribution of opioid-binding cell adhesion molecule (OBCAM) using monoclonal antibodies. Neurochem Int, 1996. 28(4): p. 373-9.

13. Miyata, S., et al., Biochemical and ultrastructural analyses of IgLON cell adhesion molecules, Kilon and OBCAM in the rat brain. Neuroscience, 2003. 117(3): p. 645-58.

14. Lodge, A.P., et al., Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system. Brain Res Mol Brain Res, 2000. 82(1-2): p. 84-94.

15. McNamee, C.J., et al., Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth. J Neurochem, 2002. 80(6): p. 941-8.

16. Li, P., et al., Postnatal expression profile of OBCAM implies its involvement in visual cortex development and plasticity. Cereb Cortex, 2006. 16(2): p. 291-9.

17. Vincenti, M.P. and C.E. Brinckerhoff, Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-1beta. Arthritis Res, 2001. 3(6): p. 381-8.

18. Reed, J., et al., Diglons are heterodimeric proteins composed of IgLON subunits, and Diglon-CO inhibits neurite outgrowth from cerebellar granule cells. J Cell Sci, 2004. 117(Pt 17): p. 3961-73.

19. Ntougkos, E., et al., The IgLON family in epithelial ovarian cancer: expression profiles and clinicopathologic correlates. Clin Cancer Res, 2005. 11(16): p. 5764-8.

20. Anglim, P.P., et al., Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer. Mol Cancer, 2008. 7: p. 62.

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21. Reed, J.E., et al., Expression of cellular adhesion molecule 'OPCML' is down-regulated in gliomas and other brain tumours. Neuropathol Appl Neurobiol, 2007. 33(1): p. 77-85.

22. Ye, F., et al., OPCML gene promoter methylation and gene expression in tumor and stroma cells of invasive cervical carcinoma. Cancer Invest, 2008. 26(6): p. 569-74.

23. Chen, J., et al., The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell, 2003. 4(5): p. 405-13.

24. Itoh, N. and D.M. Ornitz, Evolution of the Fgf and Fgfr gene families. Trends Genet, 2004. 20(11): p. 563-9.

25. Popovici, C., et al., An evolutionary history of the FGF superfamily. Bioessays, 2005. 27(8): p. 849-57.

26. Bottcher, R.T. and C. Niehrs, Fibroblast growth factor signaling during early vertebrate development. Endocr Rev, 2005. 26(1): p. 63-77.

27. Powers, C.J., S.W. McLeskey, and A. Wellstein, Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer, 2000. 7(3): p. 165-97.

28. Basilico, C. and D. Moscatelli, The FGF family of growth factors and oncogenes. Adv Cancer Res, 1992. 59: p. 115-65.

29. Burgess, W.H. and T. Maciag, The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem, 1989. 58: p. 575-606.

30. Eswarakumar, V.P., I. Lax, and J. Schlessinger, Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev, 2005. 16(2): p. 139-49.

31. Rapraeger, A.C., A. Krufka, and B.B. Olwin, Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science, 1991. 252(5013): p. 1705-8.

32. Yayon, A., et al., Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell, 1991. 64(4): p. 841-8.

33. Gotoh, N., Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins. Cancer Sci, 2008. 99(7): p. 1319-25.

34. Fujimoto, J., et al., Expression of basic fibroblast growth factor and its mRNA in advanced ovarian cancers. Eur J Gynaecol Oncol, 1997. 18(5): p. 349-52.

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37. Le Page, C., et al., From gene profiling to diagnostic markers: IL-18 and FGF-2 complement CA125 as serum-based markers in epithelial ovarian cancer. Int J Cancer, 2006. 118(7): p. 1750-8.

38. Katoh, M., Cancer genomics and genetics of FGFR2 (Review). Int J Oncol, 2008. 33(2): p. 233-7.

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Section D: Curriculum Vitae of Fellowship Applicant (To be completed by Applicant in Arial pt 11)

Please complete each section, expanding as necessary to a maximum of 2 pages not including publications. 1) Name Evangelos Ntougkos 2) Title Dr

3) Nationality Greek

4) Previous posts held (most recent first, include: dates, position, and university / institution) May 2006 - May 2008 – Postdoctoral Research Associate – Hellenic Pasteur Institute

5) Education / training (most recent first, include: dates, degree, subject, and university / institution) October 2002 - January 2006 – PhD – University of Edinburgh October 2001 - September 2002 – MSc by Research in Life Sciences – awarded with distinction – University of Edinburgh September 1998 - June 2001 – BSc (Hons) in Human Genetics – Class I – University of Leeds

6) Summary of scientific career (include key achievements and expertise relevant to the application, prizes, and any external funding that you’ve obtained independently) My scientific career began at the University of Leeds, where I first came in contact with the principles of scientific research. Upon graduation I was awarded the Alistair Stewart prize for outstanding performance in the field of human genetics. I then pursued postgraduate studies choosing a Master’s by research degree, as even at this early stage I knew I wanted to dedicate myself to research. After successful completion of projects that gave me the opportunity to experience working in different laboratories, I began my PhD project under the supervision of the sponsor of this application. My PhD thesis focused on the same field of research as the proposed project, namely the role of OPCML in epithelial ovarian cancer. Hence, I have expertise in both the theoretical background and the experimental approaches that we propose. Moreover, the sponsor and I have published together in this specific area and participated jointly in abstracts for conferences presented in the UK and abroad.

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7) Publications (Provide full references including the titles, where possible include web-links) Ntougkos, E., Rush, R., Scott, D., Frankenberg, T., Gabra, H., Smyth, J.F. and Sellar, G.C. The IgLON family in epithelial ovarian cancer: expression profiles and clinicopathological correlates. Clin. Cancer Res. 2005. 11: 5764-5768

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Section E: Recommendation from the Applicant’s current institution (To be completed by Applicant’s current supervisor in Arial point 11)

Please provide an assessment of the applicant’s abilities (up to 500 words) In April 2006, Evangelos joined my group and started his post-doctoral studies. The aim of his work was to construct fully human antibody fragments from previously isolated Fab antibody fragments, express them in mammalian cells and study their function. Evangelos is an excellent young researcher with great promise. He has confidence in his abilities and in the results that he produces. He is bright, highly motivated, enthusiastic and independent. He is constantly thinking of new experiments and is remarkably effective at transforming his ideas into results. He is an outstanding colleague. Evangelos is extremely generous with his ideas, criticisms, reagents and technical assistance and attracts younger people as a magnet. He is able to work well either independently or as part of a team, as the occasion demands. He integrates well and is always willing to assist others. Evangelos is one of the brightest young researchers I have ever known during my time at Hellenic Pasteur Institute. His knowledge and understanding of the literature was always impressive. Evangelos has a great aptitude for presentations. His written reports were always well composed and the oral presentations, either formal or informal talks, were clearly delivered and easy to follow and questions ably answered. With regard to publications arising from his work in my laboratory, we are currently carrying out some additional experiments before submitting a manuscript for review in the very near future.

In closing, I support Evangelos’ application for the Junior Research Fellowship with great pleasure and without any reservation. Name Avgi Mamalaki Title/Position Dr/Research Director Email amamalaki@pasteur.gr Telephone 30-210-6478838 Signature** Date 26/11/2008

Institutional Authorisation (i.e. Institutional stamp)

** I certify that the statements herein are true, complete and accurate to the best of my knowledge.

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Section F: Sponsor Details (To be completed by the Sponsor in Arial point 11)

Please complete each section, expanding as necessary to a maximum of 2 pages including publications. 1) Name Hani Gabra

2) Title Professor of Medical Oncology, Head of the Section of Molecular Therapeutics, Director of the Ovarian Cancer Action Research Centre, Department of Oncology

3) Tenure status Of Sponsor Tenured 4) Previous posts held (most recent first, include: dates, position, and university / institution) Current post since October 2003 April 1998-September 2003: CR-UK Laboratory Head/CR-UK Clinical Scientist, CR-UK Medical Oncology Unit, Western General Hospital Edinburgh, Honorary Clinical Senior Lecturer in Medical Oncology, University of Edinburgh August 1988-April 1998: SHO Rotation in Medicine, then successive ICRF Registrar, ICRF Clinical Research Fellow, ICRF Senior Registrar in Medical Oncology, Western General Hospital Edinburgh August 1987-August 1988: House officer posts Western Infirmary and Southern General Hospitals Glasgow 5) Recent relevant publications over the past 5 years (Provide full references including the titles, where possible include web-links) OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer.. Sellar GC, Watt KP, Rabiasz GJ, Stronach EA, Li L, Miller EP, Massie CE, Miller J, Contreras-Moreira B, Scott D, Brown I, Williams AR, Bates PA, Smyth JF, Gabra H Nature Genetics (2003) Jul;34(3):337-43. Identification of Clinically Relevant Genes on Chromosome 11 in a Functional Model of Ovarian Cancer Tumor Suppression. Stronach EA, Sellar GC, Blenkiron C, Rabiasz GJ, Taylor KJ, Miller EP, Massie CE, Al-Nafussi A, Smyth JF, Porteous DJ, Gabra H. Cancer Res. (2003) Dec 15;63(24):8648-8655 The IgLON family in epithelial ovarian cancer: expression profiles and clinicopathological correlates Ntougkos E, Rush R, Scott D, Frankenberg T, Gabra H, Smyth JF, Sellar GC. Clin Cancer Res. 2005 Aug 15;11(16):5764-8 CpG island methylation of DNA damage response genes in advanced ovarian cancer. Teodoridis JM, Hall J, Marsh S, Kannall HD, Smyth C, Curto J, Siddiqui N, Gabra H, McLeod HL, Strathdee G, Brown R. Cancer Res. 2005 Oct 1;65(19):8961-7.

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Evaluation of an inflammation-based prognostic score in patients with advanced ovarian cancer. Sharma R, Hook J, Kumar M, Gabra H. Eur J Cancer. 2008 Jan;44(2):251-6.

6) Please provide evidence of any previous mentorship over the past 5 years (this could include career path/destinations of previous lab members) Developed the post and mentored Dr Adam Paige as Non-clinical Lecturer in the department Developed the post and mentored Dr Sarah Blagden as Clinical Senior Lecturer in the department Developed the post and mentored Dr Sadaf Ghaem-Maghami as Clinical Senior Lecturer in the department Developed the post and mentored Dr Roshan Agarwal as Clinical Senior Lecturer in the department Developed and mentored Dr Euan Stronach into an OCA Senior Postdoctoral Fellowship and Junior Group Leader position in the department 7) Recommendation of Applicant (an assessment of the applicant’s abilities, under 500 words) I have successfully supervised many PhD students. Without doubt Evangelos Ntougkos is the brightest and most insightful graduate student that I have supervised. He has an excellent balance of breadth and focus and has high intellect as well as a genuine love of science. I tried very hard to get him to join us at Imperial after his PhD but he was adamant that he wished to return to Greece for personal reasons. Evangelos was very unusual in that he had a highly independent streak from the outset of his PhD and would always trust his own instincts and judgement. This led to creative solutions to intractable problems during his excellent PhD. I have now convinced him that he should apply for the JRF, which is a perfect position for someone of his real independence of mind who can be allowed to develop within the highly supportive environment that I will provide for him. I believe that without doubt this fellowship will be his making. Since he completed his PhD the OPCML field has moved on and our exciting data includes the project that he proposes. I truly believe that he should be given the opportunity to contribute to this hugely exciting area of cancer biology, and I have no doubt that he will make a major contribution.