SCIENTIFIC REPORT 2013 cruk.org SCIENTIFIC Cover image Graduate student Loic Fort from Laura Machesky’s group. REPORT 2013 BEATSON INSTITUTE CONTENTS

SECTION 1 ADVANCED TECHNOLOGIES INTRODUCTION 04 Kurt Anderson 48 RESEARCH HIGHLIGHTS 06 Beatson Advanced Imaging Resource (BAIR) BACKGROUND 10 Gabriela Kalna 49 Bioinformatics and Computational Biology CANCER RESEARCH UK BEATSON INSTITUTE Gillian Mackay 50 Metabolomics REGULATION OF CANCER CELL GROWTH METABOLISM AND SURVIVAL Nick Morrice 51 Eyal Gottlieb 12 Proteomics and Mass Spectrometry Apoptosis and Tumour Metabolism Emma Shanks 52 Danny Huang 14 RNAi Screening Ubiquitin Signalling Karen Blyth 53 Hing Leung 16 Transgenic Models of Cancer Prostate Cancer Biology Douglas Strathdee 54 Kevin Ryan 18 Transgenic Technology Tumour Cell Death Karen Vousden 20 SECTION 2 Tumour Suppression BEATSON ASSOCIATES Peter D. Adams 56 REGULATION OF CANCER CELL INVASION Epigenetics of Cancer and Ageing AND METASTASIS Daniel J. Murphy 58 Kurt Anderson 24 Oncogene-Induced Vulnerabilities Tumour Cell Migration Stephen Tait 60 Jeff Evans 26 Mitochondria and Cell Death Translational Cancer Therapeutics Robert Insall 28 UNIVERSITY OF GLASGOW Cell Migration and Chemotaxis Jeff Evans 64 Laura Machesky 30 Institute of Cancer Sciences Migration, Invasion and Metastasis Jim Norman 32 SECTION 3 Integrin Cell Biology RESEARCH FACILITIES 72 The Beatson Institute for Cancer Michael Olson 34 PUBLICATIONS 76 Research building Molecular Cell Biology CONFERENCES AND WORKSHOPS 90 Owen Sansom 36 SEMINARS 92 Colorectal Cancer and Wnt Signalling STUDENTSHIPS AND POSTDOCTORAL 94 FELLOWSHIPS Marcos Vidal 38 ADMINISTRATION 95 Drosophila Approaches to Cancer THANKS FOR SUPPORTING US 96 Sara Zanivan 40 PATRONS AND BOARD OF GOVERNORS 99 Vascular Proteomics CONTACT DETAILS 100

DRUG DISCOVERY Martin Drysdale 44 Drug Discovery Programme

2 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE 3 INTRODUCTION

Members of the Beatson Cancer This year was an extremely important and busy one for us with Metabolism Research Unit at quinquennial reviews of both the Institute and the Drug work (CaMeRU) Discovery Programme being held in June. We also took a lead role in the renewal of the CRUK Glasgow Centre.

Our Institute review was a great success and a tumour types and we were delighted to be Professor Karen Vousden fantastic endorsement of everyone’s hard work awarded a substantial increase in the funding CBE, FRS, FRSE, FMedSci over the past few years. The review panel was available to support these areas. The Centre also Director of The Beatson very positive about our progress to date and had the pleasure of welcoming Stan Kaye as Institute for Cancer Research future plans – especially our move to develop Chair of its Governance Board. Stan, who has cancer metabolism. An increasing number of worked at the Institute of Cancer Research in our research groups are doing excellent work in London for many years, brings considerable this area as can be seen from our research expertise to this role as well as extensive highlights, which include a number of knowledge of cancer research in Glasgow from metabolism-focused papers. We are also his time here as Chair of Medical Oncology. putting considerable effort into establishing our Cancer Metabolism Research Unit (CaMeRU), This year saw the departure of a long-standing headed by Eyal Gottlieb, and we have been member of the Institute David Gillespie, who actively engaged in recruiting the people and secured a position at the University of Tenerife building the resources in support of this. Our where he will continue his research programme first group leader appointment will be Jurre on checkpoints and cell cycle control. Dave Kamphorst, whose expertise is in lipid joined the Beatson in 1989 and made important metabolism and who will join us as a Beatson contributions to the work of the Institute as well departing postdocs winning fellowships and coming years. Our students had the great Associate in early 2014. as acting as a mentor and overseeing our taking up independent group leader positions pleasure of playing host to Sir Paul Nurse who postdoctoral training programme. We wish elsewhere, including Celia Berkers (Utrecht), visited this year in his role as President of the The review of the Drug Discovery Programme, Dave all the best in sunnier climes. Julia Cordero (Glasgow), Jason King Royal Society. the first since its inception, was also (Sheffield),Patricia Muller (MRC Toxicology encouraging about what the unit has achieved We also said farewell to our Laboratory Manager Unit, Leicester) and Tobias Zech (Liverpool). Finally, as every year we would like to extend our so far and its future plans. There is a growing Robert MacFarlane, who retired from the heartfelt thanks to all the donors and supporters integration between the work of the unit and Institute at the end of August after 42 years of We also saw a large number of students whose generosity makes our work possible. the rest of the Institute, an initiative that is being service. His contribution to the Institute was successfully graduate this year and we look led by the head of the DDP, Martin Drysdale. extraordinary and we wish him the very best for forward to seeing their careers develop in the his retirement. Meanwhile, Laura Bence joined We were also co-applicants, along with us to take on this key role. colleagues from the Institute of Cancer Sciences at the University of Glasgow, on a I was delighted to be elected to the American successful application to renew our CRUK Association for the Advancement of Science, Centre status. The new award includes an while one of our junior group leaders Marcos important training component in support of Vidal was recognised by being elected to the students and clinical fellows but particularly Young Academy of the Royal Society of focuses on providing infrastructure in the areas Edinburgh. of co-clinical trials, informatics, biomarkers, biospecimens and functional screens. The goal The Institute review panel highlighted the is to provide a platform of technical expertise excellent training environment we provide for and technological capabilities to support students and postdoctoral scientists and this members of the Centre across all disciplines and was reflected again this year with several of our

4 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE INTRODUCTION 5 RESEARCH HIGHLIGHTS

This section features some of the key research findings It is known that adenosine accumulates under conditions of cellular stress including during made by scientists at the Beatson Institute and Institute of tumour development and this study provides a Cancer Sciences in the past year. mechanism that links this change in metabolism to the induction of cell death via the tumour suppressor p53. The authors use microarray Cheung EC, Athineos D, Lee P, Ridgway RA, Oncogene-induced senescence (OIS) blocks data to identify A2B, the adenosine receptor, as Lambie W, Nixon C, Strathdee D, Blyth K, cancer development by acting as a brake and a p53 target gene. They go on to show how A2B Sansom OJ, Vousden KH. stopping cell proliferation. However, little is is upregulated by p53 and then activates TIGAR is required for efficient intestinal known about the metabolism involved in this apoptosis upon adenosine binding. regeneration and tumorigenesis. Dev Cell process. This study uses metabolic profiling and 2013; 25: 463-77 functional perturbations to show that pyruvate Lourenco FC, Munro J, Brown J, Cordero J, dehydrogenase (PDH), an enzyme that ties Stefanatos R, Strathdee K, Orange C, Feller SM, In this study, the authors generate a TIGAR- glucose metabolism to oxidative Sansom OJ, Vidal M, Murray GI, Olson MF. deficient mouse and demonstrate the phosphorylation, is a crucial mediator of OIS in Reduced LIMK2 expression in colorectal cancer importance of this fructose-2,6-bisphosphatase melanoma induced by the oncogene reflects its role in limiting stem cell proliferation. in regulating glucose metabolism during BRAF(V600E). Furthermore, induction of PDH Gut 2014; 63: 480-93 intestinal regeneration and adenoma activity blocks melanoma growth and development. They conclude that TIGAR progression in vivo, leading the authors to In this work, the authors demonstrate that a contributes to both tissue regeneration and suggest that the relationship between OIS and reduction in LIM kinase 2 expression in tumour development and thus could be a PDH might be exploited therapeutically. colorectal cancer is associated with shorter potential therapeutic target in diseases such as patient survival. They also show, using ulcerative colitis and intestinal cancer. Klejnot M, Gabrielsen M, Cameron J, genetically modified Drosophila and mouse Mleczak A, Talapatra SK, Kozielski F, Pannifer models, that LIM kinase 2 can constrain Dou H, Buetow L, Sibbet GJ, Cameron K, A, Olson MF. gastrointestinal stem cell proliferation and thus Huang DT. Analysis of the human cofilin 1 structure reveals colon tumour development. Essentiality of a non-RING element in priming conformational changes required for actin donor ubiquitin for catalysis by a monomeric binding. Acta Crystallogr D Biol Crystallogr Ma Y, Li A, Faller WJ, Libertini S, Fiorito F, ROS production and NF-kappaB activation E3. Nat Struct Mol Biol 2013; 20: 982-6 2013; 69: 1780-8 Gillespie DA, Sansom OJ, Yamashiro S, triggered by RAC1 facilitate WNT-driven Machesky LM. intestinal stem cell proliferation and colorectal This paper describes the crystal structure of a In this paper, the authors determine the crystal Fascin 1 is transiently expressed in mouse cancer initiation. Cell Stem Cell 2013; 12: 761-73 monomeric RING E3 ligase. RING E3 ligases structure of a C147A point mutant of human melanoblasts during development and transfer ubiquitin from E2 to substrate during cofilin 1, a protein that disassembles actin promotes migration and proliferation. In this paper, the authors demonstrate that the ubiquitination, a process that regulates protein filaments and contributes to remodelling of the Development 2013; 140: 2203-11 GTPase RAC1 is a critical mediator of degradation and which is important for many actin cytoskeleton. Actin remodelling is crucial tumourigenesis following APC loss in colorectal cellular activities. Based on their analysis of the to several processes within the cell, including This study investigates the role of fascin 1, an cancer. RAC1 is shown to be responsible for the structure, the authors suggest that interactions some such as motility and division that have a actin-bundling protein, in melanocytes and expansion of intestinal stem cells and progenitor outside the canonical RING domain are crucial role in cancer. This study is the first to describe melanoma cells in vivo and in skin explants. The hyperproliferation and thus intestinal for optimising ubiquitin transfer in both the crystal structure of an animal cofilin and authors show that fascin 1 knockout leads to transformation. Crucially, it does this by monomeric and dimeric RING E3s. reveals how its actin-binding helix undergoes a both migration and proliferation defects in triggering ROS production and NF-κB signalling. conformational change that increases the melanoblasts, the melanocyte precursors, and Kaplon J, Zheng L, Meissl K, Chaneton B, number of potential hydrogen bonds available in melanoma cells. They conclude that fascin 1 Park L, Thomason PA, Zech T, King JS, Veltman Selivanov VA, Mackay G, van der Burg SH, for substrate binding. has a role in cell cycle progression as well as DM, Carnell M, Ura S, Machesky LM, Insall RH. Verdegaal EM, Cascante M, Shlomi T, Gottlieb migration and that its expression may be Cyclical action of the WASH complex: FAM21 E*, Peeper DS*. Long JS, Crighton D, O’Prey J, Mackay G, advantageous in tumours. and capping protein drive WASH recycling, not A key role for mitochondrial gatekeeper Zheng L, Palmer TM, Gottlieb E, Ryan KM. initial recruitment. Dev Cell 2013; 24: 169-81 pyruvate dehydrogenase in oncogene-induced Extracellular adenosine sensing - a metabolic Myant KB, Cammareri P, McGhee EJ, Ridgway senescence. Nature 2013; 498: 109-12 cell death priming mechanism downstream of RA, Huels DJ, Cordero JB, Schwitalla S, Kalna In this study, the authors use Dictyostelium to *corresponding authors p53. Mol Cell 2013; 50: 394-406 G, Ogg EL, Athineos D, Timpson P, Vidal M, generate mutants of each of the five subunits of Murray GI, Greten FR, Anderson KI, Sansom OJ. the regulatory WASH complex. This allows them

6 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH HIGHLIGHTS 7 RESEARCH HIGHLIGHTS (CONTINUED)

to investigate the functions of each subunit stratification for targeted therapies and N-WASP- and FAK-dependent invasion. Ivanov A, Pawlikowski J, Manoharan I, van during actin polymerisation, lyosomal trafficking chemopreventive interventions. Curr Biol 2013 ; 23: 107-17 Tuyn J, Nelson DM, Rai TS, Shah PP, Hewitt G, and exocytosis, and highlights a distinct role for Korolchuk VI, Passos JF, Wu H, Berger SL, FAM21 in this latter step as well as in the Rajan P, Sudbery IM, Villasevil ME, Mui E, This study describes the contrasting roles of the Adams PD. recycling of the WASH complex itself. Fleming J, Davis M, Ahmad I, Edwards J, Scar/WAVE regulatory complex (WRC) and the Lysosome-mediated processing of chromatin in Sansom OJ, Sims D, Ponting CP, Heger A, Arp2/3 activator N-WASP in 3D cell migration. senescence. J Cell Biol 2013; 202: 129-43 Patel R, Gao M, Ahmad I, Fleming J, Singh LB, McMenemin RM, Pedley ID, Leung HY. The authors show that N-WASP, in cooperation Rai TS, McKie AB, Seywright M, Barnetson RJ, Next-generation Sequencing of Advanced with FAK, is pro-invasive, while WRC inhibits This study tracks cytoplasmic chromatin Edwards J, Sansom OJ, Leung HY. Prostate Cancer Treated with Androgen- invasive migration. This is contrary to its role in fragments to demonstrate that senescent cells Sprouty2, PTEN, and PP2A interact to regulate deprivation Therapy. Eur Urol published online 2D migration. WRC loss also promotes FAK- process their chromatin via an autophagy/ prostate cancer progression. J Clin Invest 2013; 14 Aug 2013, doi: 10.1016/j.eururo.2013.08.011 dependent cell transformation and tumour lysosomal pathway. This leads to a depletion 123: 1157-75 growth in vivo. The authors conclude that more of histones and is associated with tumour This work focuses on castration-resistant work is needed to determine the potential suppression. The authors suggest that This integrative study uses preclinical and prostate cancer, the mechanisms underlying significance of WRC loss in cancer. processing chromatin in this way might clinical resources to address the biology which are not fully understood. It is the first contribute to the stability of cellular underlying collaborative tumour suppressor report on the RNA sequencing of matched Trinidad AG, Muller PA, Cuellar J, Klejnot M, senescence and tumour suppression. functions. The authors identify SPRY2, PTEN prostate cancer tissue from patients before and Nobis M, Valpuesta JM, Vousden KH. and PP2A deficiencies as prognostic biomarkers after androgen-deprivation therapy (ADT) and Interaction of p53 with the CCT complex that cooperate to drive aggressive prostate highlights some ADT regulated signalling promotes protein folding and wild-type p53 cancer. This biomarker trio may facilitate patient pathways that may be targets for the treatment activity. Mol Cell 2013; 50: 805-17 of castration-resistant prostate cancer. This paper investigates the control of p53 by the Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, chaperonin CCT. The authors show that CCT Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, binds wild-type p53 and is required for correct Ridgway R, Adams PD, Anderson KI, Gottlieb E, folding of the protein. Furthermore, CCT Sansom OJ, Ryan KM. binding can regulate p53’s growth suppressive p53 status determines the role of autophagy in and invasive behaviours, each of which the pancreatic tumour development. Nature 2013; authors suggest may be differentially regulated. 504: 296-300 Institute of Cancer Sciences In this paper, the authors provide important Cruickshanks HA, McBryan T, Nelson DM, insights into the role of autophagy, a key Vanderkraats ND, Shah PP, van Tuyn J, Singh Rai regulator of cellular homeostasis, in cancer. T, Brock C, Donahue G, Dunican DS, Drotar ME, Using a mouse model of pancreatic cancer, the Meehan RR, Edwards JR, Berger SL, Adams PD. authors show that while loss of autophagy Senescent cells harbour features of the cancer blocks tumour formation in mice containing epigenome. Nat Cell Biol 2013; 15: 1495-506 oncogenic Kras, this is not the case in mice containing oncogenic Kras but also lacking p53. In this paper, the authors use genome-wide In fact, in this scenario loss of autophagy sequencing to investigate the pattern of DNA actually accelerates tumour formation, fuelled methylation in senescent cells. They observe by increased glucose uptake and metabolism. widespread hypomethylation and focal The authors note that their findings have hypermethylation similar to that seen in cancer potential implications for autophagy inhibition cells. These features are also largely retained in cancer therapy. when cells bypass senescence. The authors speculate that if these senescent cells escape Tang H, Li A, Bi J, Veltman DM, Zech T, the proliferative barrier, they might thus be Spence HJ, Yu X, Timpson P, Insall RH, already primed for malignancy. Frame MC, Machesky LM. Loss of Scar/WAVE complex promotes

8 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH HIGHLIGHTS 9 BACKGROUND

The name Beatson used in our title is in recognition of the early work of Sir George Beatson who in 1912 established a research department at the cancer hospital in Glasgow. This REGULATION department became independent from the hospital in 1967 when The Beatson Institute for Cancer Research was founded by the then Director, Dr John Paul. Dr Paul also raised sufficient OF CANCER funds to move the Institute in 1976 to our present location at Garscube Estate in Glasgow. CELL GROWTH In 1990 Glasgow University researchers moved to adjacent refitted accommodation. More recently, other teams with University affiliations have moved here to share laboratory METABOLISM facilities with us and, in 2013, to the adjoining Wolfson Wohl Cancer Research Centre. The resulting Institute of Cancer Sciences provides a cutting edge research environment AND SURVIVAL situated in the beautiful, leafy green Garscube Estate on the north-western edge of Glasgow.

Sir George Beatson 1848 - 1933 CANCER RESEARCH UK The Beatson Institute for Cancer Research BEATSON INSTITUTE

Eyal Gottlieb - Apoptosis and Tumour Metabolism Danny Huang - Ubiquitin Signalling Hing Leung - Prostate Cancer Biology Kevin Ryan - Tumour Cell Death Karen Vousden - Tumour Suppression

10 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE REGULATION OF CANCER CELL GROWTH METABOLISM AND SURVIVAL 11 deletion of kidney FH were generated by APOPTOSIS AND TUMOUR METABOLISM crossing the Fh1fl/fl mice with an appropriate Cre www.beatson.gla.ac.uk/eyal_gottlieb transgenic mouse. These mice developed multiple benign renal cysts. In order to investigate the metabolic signature associated with the loss of FH in these mice, urine was collected and analysed by LC-MS for metabolic profiling. The metabolites that consistently contributed to the urine metabolic signature of these mice were urobilin, fumarate and argininosuccinate. The presence of urobilin (the urine form of bilirubin) in the urine of mice with An early hallmark of cancer tissues is metabolic Fh1-deficient cysts strongly supports our previous report that heme degradation is reprogramming, first noted by Otto Warburg who upregulated in FH-deficient tumours (Frezzaet found that cancer cells rely on glycolysis under aerobic al., Nature 2011; 477: 225). The presence of fumarate in the urine was not unexpected conditions (the Warburg Effect). More recent research considering the high levels of fumarate detected showed that metabolic alterations in cancer include many in FH-deficient cells and tumours. However, the detection of high levels of argininosuccinate in additional pathways, and this potentially increases the the urine of mice with FH-deficient kidney cysts number of clinical targets. In fact, most, if not all tumour was somewhat surprising as this is a urea cycle Group Leader suppressors and oncogenes regulate metabolism. Our major metabolite. Argininosuccinate was also Eyal Gottlieb detected at high levels in the spent media of interest is in metabolic enzymes that also function as tumour Figure 1 patient-derived FH-deficient renal cancer cells -/- Associate Scientist OIS is accompanied by enhanced TCA cycle flux mediated by PDH activation. and in the immortalised Fh1 mouse epithelial Zachary Schug3 suppressors or oncogenes, or regulate the essential metabolic Metabolic alterations in OIS versus cycling human diploid fibroblasts were studied using a kidney cells. requirements of cancer cells. Over the past few years, my lab mass-balance model. Measured metabolic fluxes were used for balancing the delineated Research Scientists metabolic network. The biosynthetic constraints and the rates of reactions that produce Argininosuccinate is a urea cycle intermediate Simone Cardaci1 has generated state-of-the-art metabolomics capabilities for reducing equivalents NAD(P)H and FADH2 (glycolysis, pentose phosphate pathway, malic Nadja Pfetzer2 enzyme and TCA cycle) were balanced with those that oxidise them (oxygen consumption, synthesised by argininosuccinate synthase (ASS) Saverio Tardito4 studying metabolic transformations and identifying metabolic lactate dehydrogenase and fatty acid biosynthesis). The calculated reaction rates are and is subsequently metabolised to fumarate Liang (Leon) Zheng presented as coloured arrows where the thickness of the arrows represents the rate of and arginine by argininosuccinate lyase (ASL) vulnerabilities of cancer. reaction and the colour represents the ratio between OIS and cycling cells. All reactions are (Fig. 2). Interestingly, argininosuccinate aciduria 6 Scientific Officer calculated as nmol/(10 cells x hour). is observed in Inborn Error of Metabolism Elaine MacKenzie patients deficient in ASL. Therefore, it seemed Role of oxidative metabolism in consequences of perturbing the balance plausible that the increase in argininosuccinate Clinical Research Fellow oncogene-induced senescence between glycolysis and oxidative Stefan Nowicki5 results from ASL inhibition by high levels of Several tumour-suppressing mechanisms have phosphorylation on OIS and tumourigenesis, fumarate in FH-deficient cells. To test this evolved that avert the hazard of malignant and further identified pyruvate dehydrogenase Graduate Students possibility we incubated cells with uniformly Barbara Chaneton transformation sparked by oncogenic events. kinase 1 (PDK1), the inhibitor of PDH, as a labelled [13C ]-glutamine and using LC-MS Laura Galbraith One such programme is the cessation of cell vulnerable target in BRAF mutant melanoma 5 traced heavy carbon (13C) incorporation into proliferation, termed oncogene-induced cells. The inhibition of PDK1 (and the 1 FEBS TCA cycle and other intracellular metabolites. senescence (OIS). In recent years, a large body consequent activation of PDH) induced 2 Janssen Pharmaceutica Unexpectedly, argininosuccinate was quickly of evidence has shown that OIS is a senescence in established BRAF-driven 13 3 labelled with four glutamine derived C CRUK Discovery pathophysiologic mechanism that halts melanomas resulted in tumour regression in Committee/Astra Zeneca carbons, demonstrating that, in FH-deficient progression to cancer in model organisms and vivo (Kaplon et al., Nature 2013; 498: 109). 4 Italian Association for cells, argininosuccinate is not produced from humans alike. Indeed, senescence biomarkers Cancer Research citrulline and aspartate by ASS (as is the case in 5 have been reported for a plethora of pre- Metabolic adaptations induced by loss of CRUK Glasgow Centre normal cells) but from arginine and fumarate via cancerous lesions including melanocytic nevi, the mitochondrial tumour suppressor a reversed ASL reaction (Fig.2). These results prostate intraepithelial neoplasia and fumarate hydratase were confirmed by the decrease in lymphomas. Although it is generally assumed Mutations in the tricarboxylic acid (TCA) cycle argininosuccinate secretion following ASL gene that senescent cells remain metabolically active, enzyme fumarate hydratase (FH) are associated silencing, and by the increased sensitivity of surprisingly few aspects of this process have with a highly malignant form of papillary and FH-deficient cells to arginine depletion (Zheng been investigated in detail. To address this need collecting duct renal cell cancer. Although the et al., Cancer Metab 2013; 1: 12). we performed a comprehensive study of the mechanisms of tumourigenesis related to FH role and regulation of metabolism in OIS. We mutations have been extensively investigated, Publications listed on page 78 used metabolic flux profiling to screen for little is known about the overall consequences metabolic changes in central carbon and that the loss of FH has on cellular metabolism. energy metabolism in OIS evoked by mutant To address this, we utilised a previously Figure 2 Argininosuccinate is produced from arginine and fumarate via reversed ASL activity. BRAF, a common driver of senescence in characterised genetically engineered mouse (A) Under normal conditions aspartate, which is derived from the TCA cycle metabolite benign lesions (Fig. 1). We demonstrated that model with a conditional gene ablation of FH oxaloacetate (OAA), reacts with citrulline to produce argininosuccinate - a reaction catalysed pyruvate oxidation in the mitochondria via (Fh1fl/fl) and primary immortalised kidney cells in by ASS. Argininosuccinate is then converted to arginine and fumarate by ASL. (B) In light of the 13 pyruvate dehydrogenase (PDH) is necessary and which Fh1 was genetically deleted ex vivo by isotopomer distribution of glutamine-derived C4-labelled argininosuccinate, this scheme sufficient for OIS. We also studied the functional adenovirus-Cre infection. Initially, mice with represents the biochemical pathway that links TCA cycle metabolites in FH-deficient cells to the urea cycle and to argininosuccinate production.

12 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE APOPTOSIS AND TUMOUR METABOLISM 13 and these mutations abrogate E3 ligase activity changes are insufficient to account for the UBIQUITIN SIGNALLING and induce cell transformation (reviewed in Kale 1400-fold enhancement in catalytic efficiency. www.beatson.gla.ac.uk/danny_huang et al., Cancer Res 2010; 70: 4789). It remains More recently we showed that phosphorylation elusive how CBLs are regulated and how these of LHR Tyr plays an additional crucial role. By mutations could contribute to oncogenicity. determining a crystal structure of phosphoTyr363-CBLB (Tyr363 is the All CBLs share a highly conserved N-terminal corresponding LHR Tyr in CBLB) bound to a SH2-containing tyrosine kinase-binding domain TKBD substrate peptide and an E2 covalently (TKBD), a linker helix region (LHR) and a RING linked to Ub (E2~Ub), we showed that the domain (Fig. 2a) followed by a variable proline- phosphoTyr-induced E2-binding surface also rich region (PRR). The TKBD mediates substrate participates in Ub binding (Fig. 2b). Notably, the specificity by binding to proteins containing phosphate moiety of the phosphoTyr363 Deregulation in the ubiquitin (Ub) pathway is often associated phosphotyrosine motifs commonly found in directly interacts with Ub’s Thr9 side chain (Fig. RTKs or tyrosine kinases, whereas the PRR 2c). These Ub interactions alone enhance CBL’s with human pathogenesis, including cancer. Our group uses recruits proteins containing an SH3 domain. The catalytic efficiency by ~200-fold, explaining the X-ray crystallography and biochemical approaches to study LHR and RING domain play central roles in massive rate enhancement upon recruiting E2s and in mediating target phosphorylation (Dou et al., Nat Struct Mol Biol the enzymes of the Ub pathway in order to understand their ubiquitination. To gain insights into the 2013; 20: 982). regulation and function. We anticipate that the knowledge regulation of CBLs, we determined three new Figure 2 crystal structures of CBL: native CBL, CBL bound CBL’s Ty371 is one of the mutational hot spots in gained from these studies will assist in the development of Phosphorylated LHR tyrosine to a TKBD substrate peptide and CBL patients with myeloid neoplasms. Our results participates in activation of phosphorylated at Tyr371 in complex with an E2 explain why Tyr371 mutants cannot adopt an selective therapeutic targets within the Ub pathway. E2~Ub. (a) Conserved CBL’s Group Leader and a TKBD substrate peptide. Our structures active CBL configuration and optimise E2~Ub N-terminal domain containing Danny Huang TKBD, LHR and RING domain. (b) and the existing structure of CBL bound to an E2 for transfer. We are currently investigating on Ubiquitin conjugation cascade processes such as cell proliferation, Structure of Tyr363 and a TKBD substrate peptide (Zheng et al., Cell how CBL mutations could alter CBL’s Research Scientists Covalent attachment of Ub involves three differentiation, migration and survival. phosphorylated CBLB bound to 2000; 102: 533) reveal dramatic conformational conformation transitions and contribute to Lori Buetow enzymes: Ub-activating enzyme (E1), Ub- Prolonged or aberrant activation of RTKs is an E2~Ub and a TKBD substrate changes in the LHR and RING domain. We oncogenicity. peptide. (c) Close-up view of Mads Gabrielsen conjugating enzyme (E2) and Ub ligase (E3) (Fig. commonly associated with cancer. CBL proteins showed that in the unphosphorylated state, CBL pTyr363-Ub and RING-Ub 1). E1 initiates the cascade by adenylating Ub’s (CBLs), CBL, CBLB and CBLC are RING E3s that adopts an auto-inhibited conformation where Mechanism of Ub transfer by RING E3 Scientific Officer interactions. All structures are Gary Sibbet C-terminus, then forming of a covalent thioester negatively regulate RTKs, tyrosine kinases and a coloured as in (a). E2 is in light its E2-binding surface on the RING domain is RING E3s recruit E2 thioesterified with Ub intermediate with it. E1 then recruits E2 and host of other proteins by promoting their blue, TKBD substrate peptide in occluded in a competitive manner to reduce E2 (E2~Ub) and substrate to facilitate the Ub Graduate Students transfers the thioesterified Ub to E2’s catalytic ubiquitination and subsequent degradation by pink and Ub is green. binding, thereby attenuating CBL’s activity. We transfer from E2 to the amino group of a Marta Klejnot cysteine. E3 plays a pivotal role in determining the proteasome or via endocytosis. found that Tyr371 phosphorylation enhances substrate lysine. How RING E3s promote Ub Gabriele Marcianò substrate fate. In general, E3 consists of an Independent of their E3 activity, CBLs also CBL’s catalytic efficiency by 1400-fold. Tyr371 transfer is unclear. We have now determined Amrita Patel E2-binding module (HECT, RING or U-box function as adaptor proteins through phosphorylation activates CBL ligase activity by two crystal structures of RING E3 bound to E2 domain) and a protein-protein interaction interactions with a variety of proteins involved in inducing dramatic LHR conformational changes UbcH5B covalently linked to Ub at its active site. domain that confers substrate specificity. With diverse biological processes. In RTK signalling that (i) enhance overall E2 binding affinity by Both structures reveal extensive non-covalent this configuration, E3 recruits E2 thioesterified cascades, CBLs act as both positive and eliminating auto-inhibition and forming a new donor Ub interactions with UbcH5B and the with Ub and substrate to promote Ub transfer negative regulators: they propagate signals phosphoTyr371-induced E2-binding surface; RING domain. Notably an additional Ub-binding from E2 to a lysine side chain on the substrate. In downstream of activated RTKs as adaptors and and (ii) place the RING domain and E2 in element outside the RING domain is crucial in humans, the Ub pathway consists of two E1s, simultaneously ubiquitinate and promote proximity of the substrate-binding site. We stabilising the Ub’s Ile36 surface (Fig. 3). In ~30-40 E2s and ~600 E3s that ubiquitinate degradation of the same RTKs as E3s. Recent showed that phosphoTyr371-induced dimeric RING E3s such as BIRC7 and RNF4, the thousands of different substrates. studies showed that CBL mutations are found in conformational transition is required for EGFR C-terminal tail of the second RING subunit acts human patients with myeloproliferative diseases ubiquitination. Together these results as this additional Ub-binding component and, The Ub pathway has emerged as the target for demonstrate how Tyr371 phosphorylation could in CBLB, phosphorylation of LHR Tyr363 creates therapeutic intervention. Velcade, a proteasome transiently switch on CBL’s ligase activity to a phosphoTyr-induced element adjacent to the Figure 1 inhibitor, is currently used for treating patients attenuate RTK signalling (Dou et al., Nat Struct RING domain that functionally mimics the Enzymatic cascade for with multiple myeloma and relapsed mantle cell Mol Biol 2012; 19: 184). dimeric RING E3 tail in stabilising Ub. Both Ub modifications. lymphoma. Recent studies demonstrated that Ub-binding components optimise kcat and Km inhibitors of NEDD8 E1 (Soucy et al., Nature Although our results showed that LHR Tyr371 for Ub transfer. These results demonstrate that 2009; 458: 732) and E2 CDC34 (Ceccarelli et al., phosphorylation could induce dramatic as well as Ub-E2, Ub-RING and E2-RING Cell 2011; 145: 1075) induced apoptosis and conformational changes to activate CBL, these interactions, an additional Ub-binding suppressed proliferation of human cancer cells, component outside the RING domain is and are currently in clinical trials. In addition, Figure 3 essential in stabilising the donor Ub several E3 inhibitors have been developed. Requirements for E2~Ub configuration, restraining the globular Ub into a Our group is interested in understanding the activation for transfer by RING closed conformation and allowing optimal regulation and mechanistic functions of E3s. An additional component positioning of Ub’s C-terminus for transfer (Dou RING E3s with particular focus on those (highlighted in yellow) outside et al., Nat Struct Mol Biol 2012; 19: 876; Dou et the RING domain (orange) is linked to cancer. required for stabilisation of Ub al., Nat Struct Mol Biol 2013; 20: 982). by CBL (a) and by the dimeric CBL proteins and receptor tyrosine RING E3s (b). Publications listed on page 79 kinase signalling Activation of receptor tyrosine kinase (RTK) signalling cascades is important for cellular

14 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE UBIQUITIN SIGNALLING 15 Figure 1 PROSTATE CANCER BIOLOGY NGS approaches to identify RBP-regulated AS in metastasis www.beatson.gla.ac.uk/hing_leung (adapted from Kalsotra & Cooper, Nat Rev Gen 2011). (A) RBPs (orange ovals) are overexpressed in metastases and bind cis- elements (orange boxes) within a number pre-mRNAs resulting in metastasis-specific AS. Computational analysis of whole transcriptome CLIP (in vivo RBP/ RNA UV crosslinking and RNA immunoprecipitation) and mRNA-Seq datasets Prostate cancer remains a global health issue and a major differentiates directly regulated AS events from secondary cause of morbidity and mortality in men worldwide. We effects. Knockdown of RBPs continue our translational research for developing new affect the metastatic phenotype. (B) Individual splicing events are treatments for prostate cancer in the context of regulated by the cooperative or antagonistic actions of RBPs, e.g. individualised medicine. Our research programme builds Sam68 and hnRNP A2/B1. accelerating prostate carcinogenesis. Once may help the design of therapies that on ongoing work on aberrant cellular signalling (including these validation experiments are completed, we maximise anti-tumour effects while androgen receptor, Sprouty2 and ERK5). In the past 12 months, can then focus on genes with potential as novel minimising host toxicity. Group Leader we initiated new projects complimentary to existing pipelines therapeutic targets. Hing Leung Androgen deprivation therapy (ADT) is the to enrich our platform for targeted therapy. We will highlight Understanding host-tumour interactions standard treatment for locally advanced and Clinical Lecturers Carcinomatosis, cancer-associated cachexia metastatic prostate cancer. We have established Imran Ahmad some of our recent findings to illustrate our progress and and multi-organ failure are common a range of in vitro and in vivo models for prostate 1 Prabhakar Rajan direction of research. developments during prostate cancer cancer and studied ADT mediated effects. We Associate Scientist progression (including treatment resistant found that prostate cancer responds to the Rachana Patel disease). Tumour cells proliferate and spread therapeutic stress upon ADT by enhanced Analysis of the transcriptome in lethal will help to delineate therapeutic strategies for by exploiting their tissue microenvironment. cytokine production, such as IL6, through HER2 Research Scientist prostate cancer ‘incurable’ prostate cancer in clinical trials. However, it remains to be tested whether receptor tyrosine kinase signalling. The Yan Zhou Many men with prostate cancer have ‘incurable’ Figure 2 IL6 as a mediator for host- tumours fuel their growth by modulating paracrine action of IL6 renders the host (locally advanced or metastatic) disease at In vivo sleeping beauty (SB) transposon Scientific Officer tumour interactions to drive other (or distant) organs. Understanding cachectic by inducing lipolysis in adipose tissue, diagnosis and a poor prognosis despite screen to identify novel genes interacting Janis Fleming tumour progression. such ‘parasitic’ modes of tumour growth resulting in elevated levels of circulating treatment. This observation may be due, in part, with Pten loss in prostate tumourigenesis cholesterol and free fatty acids. Intriguingly, Technicians/Research to the functional complexity of the cancer cell Mutations in the tumour suppressor PTEN have despite ADT, sustained prostate cancer growth Associates transcriptome primarily as a result of alternative been associated with the development and arises from self-sufficiency for androgens due to Carolyn Loveridge mRNA isoforms, fusion transcripts and progression of clinical prostate cancer. Jacqueline Stockley a combination of increased expression of non-coding RNAs. In addition to expression Transgenic mice carrying homozygous deletion steroid biosynthetic enzymes from IL6- Clinical Research Fellows changes, metastasis may be mediated by in Pten develop prostate cancer after a long mediated autocrine function and enhanced Lisa Bashford structurally or functionally differing protein latency (> 6 months) suggesting that additional cholesterol uptake required for de novo 2 Sarah Slater isoforms produced by alternative pre-mRNA genetic ‘events’ are required. We wish to intracellular synthesis of androgen (Fig. 2). In splicing (AS), controlled by RNA-binding discover the novel genetic events that Graduate Student other words, tumours use cytokines to proteins (RBPs). Our and others’ work has cooperate with Pten loss to drive prostate Elspeth Brzezinska modulate distant adipose tissue to ‘fuel’ tumour implicated interacting RBPs Sam68 and hnRNP cancer using a functional forward genetic growth and survival. Hence, our ongoing efforts 1 CRUK Clinician Scientist A2/B1 in prostate cancer, epithelial- screen in a transgenic mouse model. focus on designing therapeutic strategies to 2 MRC mesenchymal transition (EMT) and metastasis target and disrupt this communication at (Fig. 1). We are examining the role of Sam68 and In collaboration with Owen Sansom and Dave multiple levels, which may ultimately (i) decrease hnRNP A2/B1 in EMT and metastasis using Adams (Sanger Centre, Cambridge), male mice treatment resistance and metastatic progression prostate cancer cell lines, preclinical models, with the appropriate SB genotype: SB:PtenNull of the cancer and (ii) stall the detrimental effects, and primary tumours and metastases from (PB-Cre4:Ptenfl/flT2Onc3/+Uren/+) and such as cachexia, on the host. patients with prostate cancer. In particular, we littermate controls [PtenNull (PB-Cre4:Ptenfl/fl) and are interested to know how changes in SBControl (PB-Cre4:T2Onc3/+Uren/+)] were Taken together, we are making excellent expression of Sam68 and hnRNP A2/B1 proteins generated and aged to study prostate progress in our understanding of important affect the cancer cell transcriptome during tumourigenesis. SB:PtenNull mice exhibited molecular events in aggressive (and treatment prostate cancer progression. Our work uses significantly accelerated prostate resistant) prostate cancer. Through this new next generation sequencing (NGS) and will tumourigenesis, demonstrating larger tumours knowledge and our novel model systems, we enumerate RBP target mRNA isoforms in and enhanced metastasis compared to PtenNull are now in the position to consider new prostate cancer metastasis, furthering our controls. We are now studying whether the therapeutic strategies based on these events. understanding of RBP-driven metastasis. We identified transposon integration sites represent hope that mRNA targets identified in this study genes (or pathways) involved as ‘driver events’ in Publications listed on page 80

16 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE PROSTATE CANCER BIOLOGY 17 Figure 1 ABCG2 (Fig. 2). Moreover, together with TUMOUR CELL DEATH p53-mediated activation of Christine Dufès (University of Strathclyde), we A2B primes cells to die in were able to show that activation of E2F1 in www.beatson.gla.ac.uk/kevin_ryan response to accumulation of extracellular adenosine. tumour xenografts caused drug effluxin vivo Cellular stress and certain indicating that E2F1 activation in tumours could chemotherapeutic drugs cause theoretically diminish the availability and accumulation of extracellular efficacy of a systemically administered adenosine that results in cell chemotherapeutic drug. Importantly, we also death in the presence of the adenosine receptor, A2B. Since found that activation of E2F1 could markedly tumour cells often display reduce the amount of cell death caused by the altered metabolism and chemotherapeutic drug, mitoxantrone and that accumulate extracellular this effect could be reversed by inhibition of adenosine, our findings ABCG2 (Fig. 2). The aim of our group is to understand the factors represents a novel link between cancer-related of the three major drug transporters that has regulating cell viability in cancer. Since it is known that metabolic alterations and been implicated in human cancer and is p53 status switches the role of autophagy in inhibition of cell death mechanisms is a common event in tumour cell death. considered to be a mediator of drug resistance tumour development in multiple settings. Macroautophagy (hereafter autophagy) is a tumour development, this poses problems for many forms membrane trafficking process that delivers of chemotherapy that utilise cell death pathways, leading to Subsequent analysis of the relationship between cellular constituents to lysosomes for E2F1 and ABCG2 indicated that E2F1 expression degradation. It is a major homeostatic drug resistance. We are investigating both known cell death causes elevation of ABCG2 in a variety of mechanism within cells and is a very adaptable regulators as well as searching for novel proteins and primary and tumour-derived cell lines from a process responding to the needs of the cell in Group Leader pathways that control cell viability and chemosensitivity. We variety of tissues. Moreover, we were able to response to various forms of cellular stress. Kevin Ryan identify an E2F1-responsive element within Autophagy is a major regulator of cell viability envisage that the knowledge gained from our studies will be the ABCG2 promoter and chromatin and has been shown to have both oncogenic Research Scientists immunoprecipitations revealed that a site and tumour suppressive effects. Alice Baudot1 translated and lead to the improvement of existing clinical within the promoter was indeed bound by Damian Graczyk2 E2F1. Our analysis of tissue microarrays from In order to target autophagy therapeutically, we 3 regimens or new targets for therapeutic intervention. Sanket Joshi human lung cancer also revealed that there considered that it is important to know where Jaclyn Long1 Robin Macintosh4 was a highly significant correlation between and when autophagy is either oncogenic or Metabolic cell death priming downstream factors from mitochondria and programmed high E2F1 and ABCG2 levels in this very tumour suppressive in each tumour type. Scientific Officer of p53 cell death. common form of cancer. Therefore, together with Jen Morton and Owen James O’Prey The p53 tumour suppressor is a major cell death Sansom, we chose to examine the role played Figure 2 regulator. Recently, several studies have shown We considered that chemotherapeutic drugs Since ABCG2 is a multidrug transporter, we by autophagy in a mouse model of pancreatic Clinical Research Fellow E2F1 activates ABCG2 leading to Mathias Rosenfeldt 4 that p53 can also respond to and can modulate may cause activation of ADORA2B and this chemotherapaeutic drug questioned whether E2F1 could induce drug ductal adenocarcinoma (PDAC) that is cellular metabolism. We reasoned therefore that would cause cell death in situations of efflux and decreased cell death. efflux and if so, whether this effect was in a characterised by activation of oncogenic Kras. Graduate Students the ability of a cell to respond to changes in metabolic stress. To our surprise, however, we E2F1 activates expression of the concentration dependent manner. Our studies In otherwise wild-type mice, the presence of Yu Liu5 metabolism might be connected via p53 to the found that cisplatin not only causes activation of multidrug transporter ABCG2 via revealed that E2F1 could induce drug efflux in a oncogenic Kras in the pancreas led to direct promoter binding. Higher Michaela Mrschtik eradication of stressed cells by programmed ADORA2B but also causes accumulation of variety of cells and that this effect was development of PDAC over time. In the absence Pablo Sierra Gonzalez6 levels of E2F1 result in higher cell death. As a result, we searched microarray extracellular adenosine. Importantly, further levels of ABCG2, increased drug completely reversed by either chemical of autophagy, however, oncogenic Kras caused Visitor screens for genes that were activated by p53 investigation revealed that a major component efflux and reduced cell death. inhibition or RNAi-mediated knockdown of a marked increase in pre-cancerous lesions but Pepijn Schoonen and that may act as a bridge between tumour- of p53-dependent cell death in response to progression to PDAC was blocked. By contrast, associated metabolic changes and cisplatin was actually mediated via A2B upon additional deletion of p53 in the pancreas, 1 AICR programmed cell death. This analysis resulted in signalling (Fig. 1). These studies therefore tumours now not only formed in the absence of 2 EMBO the identification of the adenosine receptor, identify a new cell death pathway downstream autophagy but they appeared at an earlier time 3 until December ADORA2B as a new p53 target gene. of p53 and provide considerable insight into the when compared to autophagy proficient mice. 4 until September mechanism of action of this important The loss of p53 therefore uncovers a tumour 5 until October ADORA2B encodes A2B, which is one of four chemotherapeutic drug. suppressive role for autophagy and the tumours 6 from October human cell surface receptors that are activated in these mice exhibited enhanced glucose by adenosine, the backbone of ATP. Extracellular E2F1 mediates drug resistance via ABCG2 uptake. Metabolic analysis of tumour-derived adenosine accumulates under conditions of The E2F1 transcription factor is a regulator of cell cell lines (undertaken with the help of Gillian metabolic stress such as hypoxia and it is known cycle progression and is frequently deregulated Mackay and Eyal Gottlieb’s group), revealed that the extracellular space of many solid in human cancer. In addition to its role in cell that this enhanced glucose uptake was tumours contains unusually high levels of cycle control, E2F1 has been reported to be a accompanied by increased steady-state pools adenosine. In line with our idea that A2B might promoter of programmed cell death. To of glycolytic and pentose phosphate pathway be a metabolic stress sensor, we found that investigate E2F1 function further we performed intermediates that are known to fuel tumour treatment of A2B-expressing cells with microarrays screens to identify new E2F1 target growth. These findings therefore increase our adenosine analogues or exposure of these cells genes. To our surprise, the gene that underwent understanding of the role of autophagy in to hypoxia, resulted in a potent apoptotic the greatest fold induction upon E2F1 activation cancer and provide important consideration response. This apoptotic response involved was the ABC family transporter, ABCG2. ABCG2 for the use of autophagy modulators in downregulation of anti-apoptotic members of can efflux natural compounds and a broad cancer therapy. the Bcl-2 family permitting release of apoptotic spectrum of chemotherapeutic drugs. It is one Publications listed on page 83

18 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR CELL DEATH 19 function. We found that wild-type p53 binds to TUMOUR SUPPRESSION the chaperonin CCT and that this interaction www.beatson.gla.ac.uk/karen_vousden promotes the folding of the p53 protein. Inhibition of this interaction, by either depletion of the CCT complex or mutation of p53 to prevent binding, resulted in the expression of misfolded p53, which adopts a conformation similar to that seen in tumour derived mutants. This misfolding was accompanied by a reduction in p53-dependent gene expression and the acquisition of an ability to promote invasion. These studies show that the We have continued with our studies of how wild-type p53 interaction of p53 with the CCT complex is important to promote the tumour suppressor helps cells to adapt to metabolic stress. These functions of p53, functions of p53 and that a failure to bind CCT Figure 1 antioxidant N-acetyl L-cysteine (NAC), promotes the oncogenic activity of wild-type or p53-induced proteins such as TIGAR, make a contribution to Loss of TIGAR leads to ROS suggesting that TIGAR acts to provide accumulation and defects in p53. It therefore seems possible that one facet antioxidants and nucleosides for intestinal the response of cells to stress such as nutrient or oxygen proliferation that either reduce of the control of a normal p53 response is growth. In an intestinal adenoma model where regeneration of damaged through regulation of protein conformation and starvation, challenges that are faced by most developing intestinal epithelium (LHS) or Apc is deleted in LGR5+ intestinal stem cells, that modifications that prevent p53/CCT lower tumour burden in an mice deficient in TIGAR showed a reduction in tumour cells. We are beginning to translate these new insights interactions may allow the cell to toggle intestinal adenoma model (RHS). total tumour burden and average tumour size in Figure adapted from Cheung et between the different activities of p53. It will be into models of cancer growth in vivo, with the goal of testing the small intestine compared to wild-type mice Group Leader al., Dev Cell 2013. very interesting to determine whether the (Fig. 1). TIGAR is also highly expressed in these new therapeutic strategies. binding of p53 to CCT is a regulated step and Karen Vousden adenomas when compared to the surrounding which signals mediate this event. CBE, FRS, normal tissue, supporting the importance of Survival and adaptation to metabolic stress tumours lacking p53 developed significantly TIGAR in proliferating tissue. A similar Functions of mutant p53 FRSE, FMedSci The pro-survival functions of p53 are likely to more slowly than p53-proficient tumours under contribution of TIGAR to tumour progression In a continuation of our analysis of how mutant represent an important component of the p53 conditions of serine limitation. Our studies also was also observed in the colon and importantly, Associate Scientist p53s function to drive invasion and metastasis, response, not only in the regulation of cancer demonstrated that serine uptake supports the the decrease in tumour burden observed in Eric Cheung1 we have examined in more detail the development but also in other aspects of health Warburg effect and that a serine deficient diet TIGAR deficient mice correlated with a greater importance of the interaction of mutant p53 and disease, e.g. metabolic homeostasis, ageing significantly enhanced the survival of tumour survival in these animals. In vitro, the defective Research Scientists with the p53 family member, TAp63. Our Celia Berkers2, 5 and diabetes. Previous work showed that the bearing mice. These results therefore suggest growth of TIGAR null tumour crypts could also previous in vitro studies strongly suggested that Andreas Hock presence of wild-type p53 allows cells to survive that serine depletion may have a potential role in be rescued with antioxidants and nucleosides. Flore Kruiswijk6 the ability of mutant p53 to inhibit p63 was under conditions of glucose limitation and we anti-tumour therapy, particularly in cells lacking The pentose phosphate pathway (PPP) has been Christiaan Labuschange1, 7 important in driving the gain of function, and so 1 found over the past year that lack of p53 makes p53, and future studies will focus on examining shown to be of particular importance in redox Oliver Maddocks we carried out studies in vivo to assess the Inbal Mor1 cells more vulnerable to death in response to the effects of serine starvation on the homeostasis under hypoxic conditions, and consequences of loss of TAp63 in the context of Patricia Muller3, 8 serine depletion. development of cancer in various genetically TIGAR deficient crypts were found to be more 3, 9 loss of p53. This work showed that loss of both Antonio Trinidad modified models. sensitive to hypoxia than wild-type ones. While TAp63 and p53 recapitulated the effect of We showed that cancer cells rapidly utilise the ability of TIGAR to promote tumour Associate Clinical Scientist mutant p53 expression in promoting the exogenous serine, significant amounts of which development might appear counterintuitive to Patricia Roxburgh Functions of TIGAR in supporting tissue development of metastatic tumours. However, are used in the synthesis of glutathione and its function in the p53 tumour suppressor regeneration and cancer development the incidence of metastasis remained lower in Scientific Officer nucleotides. Serine deprivation triggered To explore the importance of TIGAR activity in pathway, it is important to note that in tumour the TAp63/p53 null tumours than in wild-type Robert Ludwig activation of the serine synthesis pathway (SSP) the context of a whole animal, we generated cells overexpressing TIGAR, expression of p53 expressing ones, indicating that mutant p53 in both wild-type p53 expressing and p53 null both constitutive and conditional deletions of TIGAR is uncoupled from p53 expression. Graduate Student also provides other functions that drive an 4 cells. In collaboration with Eyal Gottlieb’s group, TIGAR in mice. We showed that TIGAR is Indeed, closer analysis in tumour cell lines Pearl Lee invasive phenotype. Further evidence for this we found that serine is an activator of PKM2, an dispensable for normal growth and showed that the basal expression of TIGAR is model was provided by our identification of 1 ERC effect that helps to maintain glycolytic flux. development in mice but plays a key role in not dependent on the maintenance of Dicer as a target for mutant p53. Dicer is a key 2 Rubicon Reduction in intracellular serine levels resulted allowing intestinal regeneration (Fig 1). wild-type p53. The ability of a p53 target protein component in the processing of microRNAs 3 AICR in rapid suppression of aerobic glycolysis and Following ablation of the intestinal epithelium to become oncogenic when no longer properly and previous studies have shown that reduction 4 thus increased flux to the TCA cycle and SSP. through whole body irradiation or genotoxic controlled has also been described for other MRC of its activity can promote metastasis. We found 5 These initial responses to serine starvation were stress, mice deficient for TIGAR showed mediators of the p53 survival response, such as until August that the expression of mutant p53 6 not dependent on p53. However, serine reduced regenerative capacity in their intestinal carnitine palmitoyltransferase 1C. from September downregulated Dicer expression through both depletion resulted in activation of the p53-p21 crypts. Similarly, in a model of ulcerative colitis in Understanding how these genes are regulated 7 from August direct inhibition of TAp53 but also through response, and the subsequent cell cycle arrest the colon, mice that were deficient for TIGAR will be critical in determining their role in cancer 8 until June TAp63-independent mechanisms. Our studies was found to be necessary for the promotion of showed poorer recovery. As seen in cultured development. 9 until December reveal a further component of the pathway cell survival, efficiently channelling depleted cells, loss of TIGAR expression was through which mutant p53 can function and serine stores to glutathione synthesis and accompanied by an increase in ROS. Further Control of p53 conformation by CCT binding also support previous evidence showing that preserving cellular antioxidant capacity. Cells investigation using an in vitro intestinal crypt The activity of p53 and whether it functions as a mutant p53s act through TAp63-dependent and lacking p53 failed to complete the response to culture model showed that organoids lacking tumour suppressor or oncogene appears to be independent mechanisms. serine depletion, resulting in oxidative stress, TIGAR were less able to form crypt structures in determined, to some extent, by the reduced viability and severely impaired a three-dimensional tissue culture model. conformation adopted by the protein. Tumour Publications listed on page 86 proliferation. The role of p53 in supporting derived point mutations in p53 lead to the cancer cell proliferation under serine starvation These defects in TIGAR-/- cells could be rescued expression of a conformationally altered protein was translated to an in vivo model, where following the addition of nucleosides or the that has acquired invasive and metastatic

20 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR SUPPRESSION 21 REGULATION OF CANCER CELL INVASION AND METASTASIS

CANCER RESEARCH UK BEATSON INSTITUTE

Kurt Anderson - Tumour Cell Migration Jeff Evans - Translational Cancer Therapeutics Robert Insall - Cell Migration and Chemotaxis Laura Machesky - Migration, Invasion and Metastasis Jim Norman - Integrin Cell Biology Michael Olson - Molecular Cell Biology Owen Sansom - Colorectal Cancer and Wnt Signalling Marcos Vidal - Drosophila Approaches to Cancer Sara Zanivan - Vascular Proteomics

REGULATION OF CANCER CELL INVASION AND METASTASIS 23 fraction of Src active cells in the tumour core but TUMOUR CELL MIGRATION significantly reduced the fraction of those at the www.beatson.gla.ac.uk/research/kurt_anderson tumour margin, i.e. the region where local tissue invasion occurs. Finally, we examined response to dasatinib treatment as a function of proximity to vasculature (Fig. 2). We found that over a range of ~100 µm the fraction of Src active cells increased with increasing distance from the nearest blood vessel, with about 90% of cells classified as Src active over 100 µm. Dasatinib treatment significantly reduced the fraction of Src active cells at all of the distances measured, Our work is focused on development of imaging approaches Figure 2 although the effects were strongest for cells to study the cellular and molecular dynamics of metastasis nearer to blood vessels. in vitro and in vivo. Metastasis is linked to mortality in most Development of adaptive optics for deep tissue multiphoton imaging epithelial cancers. Metastatic invasion is challenging to Adaptive optics have the potential to increase study because it occurs randomly over large scales of time signal intensity and spatial resolution by sharpening the focus of a laser beam used for and space, and sensitively depends on features of the local multiphoton excitation deep within tissue. This tumour microenvironment. approach can be use to correct for aberrations Group Leader in focus introduced by both the optics of the Kurt Anderson imaging system and refractive index variations Our goal is to develop mechanistic readouts colleagues from NHS Greater Glasgow and of the sample. In collaboration with Amanda Research Scientists of cell migration and apply them to mouse Clyde, including Gerry Gillen and the clinical Wright and Keith Mathieson (Institute of Shereen Kadir cancer models including pancreatic ductal team from the Glasgow PET Centre, Jonathan Photonics, University of Strathclyde), Caroline Ross McQueenie adenocarcinoma (PDAC), melanoma and Owen from the Gartnavel Cyclotron and Sally Figure 3 Müllenbroich spent six months working with breast. We were the first group to apply Pimlott from the Radiopharmaceutical Ewan McGhee on a fellowship funded by Scientific Officer Figure 2 this we made use of a FRET reporter that Juliane Schwarz fluorescence recovery after photo-bleaching Dispensary. Our goal is to develop the use of INSPIRE to develop an adaptive optics module (FRAP) in mice to study the dynamics of the cell preclinical imaging, both as an investigative tool Intravital micrographs of PDAC changes conformation in response to for use on one of our LaVision TRIM cells expressing a Src FRET Graduate Students adhesion tumour suppressor E-cadherin in for the study of disease and response to therapy phosphorylation by active Src. reporter grown as subcutaneous microscopes. The module they developed was Orchi Anannya2 tumour cell migration. More recently we have and as an operational tool for staging disease tumours. Intensity image (left) based on a deformable mirror placed in the Zahra Erami Rud Majani demonstrated the first in vivo use of progression, in order to increase the shows PDAC cells imaged using We first measured the level of Src activity in OPO illumination pathway and imaged via a 4f Max Nobis CFP (green), collagen I imaged control and dasatinib treated PDAC cells, in Meg Pajak1 fluorescence lifetime imaging (FLIM) to study translational utility of our mouse cancer models. system into the back-focal plane of the the activation and response to therapy of the Initial studies have focused on using FDG to using SHG (magenta), and order to establish the range of fluorescence microscope objective. The mirror shape was vasculature imaged using 1 Bruker small GTPase Rho and the non-receptor measure glucose uptake and FLT to measure lifetimes associated with high and low Src Quantum Tracker 655 (red). determined using a random search algorithm 2 from October tyrosine kinase Src during mutant p53-driven proliferation, primarily in pancreatic and prostate Fluorescence lifetime image activity. This enabled us to classify individual that was optimised on the basis of signal invasion of pancreatic cancer cells. We are also tumour models. A significant milestone was (right) shows that Src activity cells as being Src active or inactive. Using this intensity from the sample. Significantly, we developing the preclinical use of PET, SPECT passed this year with the publication of our first (colour coded according to approach we found that following drug found that mirror shapes optimised using one and CT. PET data showing enhanced FDG uptake in intensity map on the left) washout, cells in culture rebounded to a high sample were transferrable to other samples, increases with increasing pancreatic tumours deficient for p53 and ATG7 level of Src activity, which peaked at 3 hours and distance from the edge of the meaning that the lengthy optimisation Figure 1 Preclinical imaging (Fig. 1). nearest blood vessel (white returned to baseline by 6 hours. A similar procedure does not need to be performed prior Merged PET and CT images We recently acquired an Albira (Bruker) tri-modal dotted line). phenomenon was observed when the same to each imaging session. Also, the signal derived showing enhanced FDG uptake in Kras G12D/+ p53 -/- mice in scanner for PET, SPECT and CT imaging of Intravital FLIM-FRET imaging of therapeutic cells were grown subcutaneously in nude mice from collagen second harmonic generation Figure 3 the absence of the essential mice, extending our imaging range from Src inactivation and subjected to 3 days of dasatinib treatment, (SHG) can be used in place of two-photon Images of mouse skin showing autophagy gene Atg7. subcellular molecular dynamics to whole body Pancreatic cancer is one of the most lethal however the rebound peak occurred at 16 hours ~2x gain in signal resulting from excited fluorescence as a signal for mirror Quantification compares uptake molecular imaging. This new work is being forms of human cancer, with an overall 5-year the use of adaptive optics. SHG and return to baseline was complete by 24 optimisation. This is advantageous because in three mice of each genotype. undertaken in close collaboration with survival rate of less than 5%. Initiating KRAS from collagen I (green) and hours. To investigate spatial aspects of Src unlike fluorescence, the second harmonic mutations occur in approximately 90% of fluorescence from Quantum activation during invasion, PDAC cells signal does not photo-bleach over time. As a human PDAC, while p53 mutations arise in Dots (red) were imaged in mouse expressing the Src FRET probe were plated onto result, signal improvements of between two and skin using either a flat (left) or 50-75% of human pancreatic cancer. Previous organotypic cultures derived from acid optimised (right) mirror shape. four times were achieved in a variety of samples, work has demonstrated that p53 mutation, extracted collagen I. After 6 days, cells had including organotypic cultures, zebrafish rather than loss, can drive metastasis in a mouse invaded over 120 µm into the matrix. embryos, and freshly excised mouse skin and model of pancreatic cancer. The metastatic Interestingly, we observed a gradient of Src intestinal tissue (Fig. 3). effect of mutant p53 in this model can be activation within the invading cells: the substantially reversed by treatment with the percentage of Src active cells increased with Publications listed on page 76 clinically approved Src inhibitor dasatinib. We increasing invasion depth. When the same cells therefore investigated spatial and temporal were grown subcutaneously, we observed there aspects of Src inactivation by dasatinib using were significantly fewer Src active cells in central fluorescence lifetime imaging of fluorescence tumour regions compared to the tumour resonance energy transfer (FLIM-FRET). To do margins. Dasatinib treatment did not alter the

24 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR CELL MIGRATION 25 with metastases compared with those treated Biological function of key tumour suppressor TRANSLATIONAL CANCER THERAPEUTICS with vehicle control. However, there was no genes and oncogenes in PDAC www.beatson.gla.ac.uk/jeff_evans improvement in survival when compared with We have also developed a number of novel vehicle control due to the morbidity of the models with a range of genetic backgrounds in primary tumour burden. Consequently, we are collaboration with Owen Sansom’s group, also exploring whether inhibition of metastases including those with targeted deletion of Pten, by dasatinib can be combined with local disease Apc, Myc or Arf. These models will help us control of the primary tumour by gemcitabine, understand the biological function of key radiation therapy or both to improve survival in tumour suppressor genes and oncogenes in the mice with locally advanced, unresectable vivo in both normal tissues and tumours. They disease but without evidence of visible will also allow us to identify and characterise the metastatic disease. Treatment with dasatinib in signalling pathways that are deregulated at the Work in our group is aimed at developing novel laboratory combination with twice-weekly injection of early stages of pancreatic cancer, during the gemcitabine resulted in a further inhibition of development and progression of the invasive models that will allow us to understand the biological metastatic spread compared with dasatinib and metastatic phenotype, and in advanced function of key tumour suppressor genes and oncogenes treatment alone (11% exhibited metastasis disease. compared with 33%). Most excitingly, however, in vivo. We aim to identify and characterise the signalling we also observed that average survival was For example, we previously demonstrated that pathways deregulated at different stages of pancreatic cancer significantly increased by over 60 days. These Lkb1 haplo-insufficiency cooperates with observations have contributed to the KrasG12D to cause PDAC. Mechanistically, we and those that are potential therapeutic targets. Using these development of a global, commercially showed that LKB1 deficient KRASG12D-induced models, we will also determine how potential anti-cancer sponsored clinical trial that aims to improve tumours exhibited reduced levels of the tumour Group Leader progression-free and overall survival in patients suppressors p53 and p21, and we proposed that agents might best be evaluated in subsequent clinical trials. G12D Jeff Evans with locally advanced disease by inhibiting the this reduction in p53 and p21 allows KRAS development of metastases. bearing cells to overcome a senescent barrier to tumour formation. Moreover, haplo- Associate Scientist Infiltrating ductal carcinoma of the pancreas improving relapse-free and overall survival Jennifer Morton Currently, we are pursuing these observations insufficiency forp21 also synergised with (PDAC) is the fifth commonest cancer and the following resection of invasive pancreatic further to determine if the administration of KrasG12D to drive PDAC. We have extended these fourth commonest cause of cancer deaths in cancer, to determine the role of intra- and Research Scientist dasatinib, as monotherapy or in combination observations to show that activation of the PI3K/ Amy Au1 the UK. Aggressive invasion and early peri-tumoural inflammation in PDAC with gemcitabine, after potentially curative Akt/mTOR pathway, which occurs in metastases are characteristic of the disease, development and progression, and to develop a resection of the localised primary tumour can approximately 20% of patients, is associated with Scientific Officer such that 90% of patients have surgically ‘personalised medicine’ approach to treatment Saadia Karim inhibit the development of metastases and poor prognosis in human PDAC patients. Loss unresectable disease at the time of diagnosis. of PDAC models from a range of genetic therefore reduce or delay disease recurrence or deficiency of Pten (and consequent 1 Furthermore, most systemic therapies are backgrounds that ultimately might influence the University of Glasgow and improve relapse-free and overall survival. activation of Akt) abrogates Ras-induced largely ineffective in advanced, inoperable management of advanced disease in the human However, the Pdx1-Cre, Z/EGFP, L SL-Kras G12D/+, senescence and leads to acceleration of PDAC disease, and the estimated 5-year overall population. L SL-Trp53 R172H/+ model develops multiple progression in laboratory models. survival is less than 5%. Gemcitabine has modest tumours throughout the pancreas making it clinical benefit and a marginal survival Evaluation of putative anti-invasive therapies difficult to accurately stage disease and achieve Currently, we are using putative inhibitors of a advantage in patients with advanced pancreatic in pancreatic cancer models therapeutically effective tumour removal. We number of pathways as clinically relevant tools cancer. Further small improvements in overall One aim of our work is to determine how are now using a number of different approaches to explore the activity of these agents in survival may be achieved with the addition of potentially anti-invasive agents might best be to address this problem, including generating laboratory models with the relevant genetic either erlotinib or capecitabine to gemcitabine used in the clinical management of epithelial an ‘inducible’ model (Pdx1-Cre-ERT, KrasG12D, background. Based on these observations, we and encouraging results have been observed in cancers. In our initial studies, we defined the p53R172H or Elastase-CreER, KrasG12D, are developing the concept of an adaptive trials with the FOLRIFINOX and gemcitabine/ mode of action of anti-cancer drugs that are p53R172H) in which we can alter the timing design ‘umbrella’ clinical trial in which patients nab-paclitaxel combination regimens. However, currently in clinical evaluation and tested the and efficiency of induction of genetic are recruited into multiple treatment arms based the median survival of patients with advanced hypothesis that these agents may have anti- modifications thus targeting a smaller number on their molecular profile. Critical to these pancreatic cancer remains poor. In addition, the migratory and hence anti-invasive and/or of cells in the adult pancreas rather than approaches will be identifying potential majority of patients who undergo potentially anti-metastatic properties. Initially, we used generating tumour promoting genetic biomarkers in murine models and confirming curative resection for small, localised lesions clinically relevant, pharmacologically active modifications in the pancreas at an embryonic the potential clinical relevance of these in inevitably develop recurrent or metastatic anti-cancer agents as experimental tools. stage as in the Pdx1-Cre, Z/EGFP, L SL-Kras G12D/+, human tissue microarrays, and developing disease, presumably due to the presence of L SL-Trp53 R172H/+ model. robust assays for patient selection to enrich the distant micro-metastases at initial diagnosis. We demonstrated that Src kinase may be a clinical trial population and to demonstrate a Adjuvant (post-operative) chemotherapy can relevant target for therapeutic intervention Having used this approach to refine the clinical biological, as well as a clinical, anti-tumour improve outcome, although overall survival following resection of PDAC and that Src activity evaluation of existing anti-cancer agents, we are effect. remains disappointing. Consequently, the is upregulated during progression to invasive now using principal component analysis, in development of more effective strategies to PDAC in the Pdx1-Cre, Z/EGFP, L SL-Kras G12D/+, collaboration with Owen Sansom and Andrew Publications listed on page 77 treat pre-invasive pancreatic cancer, micro- L SL-Trp53 R172H/+ model. We also showed that Biankin (University of Glasgow), to define a metastatic disease, and advanced disease is of treatment with dasatinib, an inhibitor of Src, in functionally relevant signature associated with paramount importance. vitro resulted in a dose-dependent inhibition of survival in resected PDAC in order to understand Src activity, and of migration and invasion of the key pathways that drive poor survival and to Our work aims to develop strategies for early PDAC cells. Furthermore, we demonstrated that identify targets for the development of novel detection of pre-invasive disease, to evaluate in vivo treatment with dasatinib from 10 weeks anti-invasive agents. putative anti-invasive therapies with the aim of of age significantly reduced the number of mice

26 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE TRANSLATIONAL CANCER THERAPEUTICS 27 We are now performing similar studies with CELL MIGRATION AND CHEMOTAXIS cancer cells. We will use high-resolution, www.beatson.gla.ac.uk/robert_insall three-dimensional microscopy determine whether human tumour cells use the same mechanism as Dictyostelium for chemotaxis and where chemotactic signals originate. Parameters such as cell speed are widely used but are broadly irrelevant in our new model. Instead, we measure the rate at which pseudopods are made and change shape, the instantaneous velocity at which pseudopods move, and the regulation of pseudopod When tumours metastasise, the regulation of cell movement retraction. We also want to identify which proteins are used to regulate tumour cell goes wrong - cancer cells invade other tissues, and spread chemotaxis and whether they are the same for through the blood and lymph systems to form secondary all metastatic cells or if not, how broad the range is. It is now clear that there are at least two tumours. This spreading behaviour is one of the most feared dissimilar mechanisms that drive tumour cell features of cancer and a principal driver of death in patients. movement. We want to know whether there Figure 1 the cellular SCAR is heavily phosphorylated but really are exactly two mechanisms or something Despite this, we do not understand what makes cells move or Cytokinesis in Dictyostelium a rare dephosphorylated form seems to be more variable. Recent experiments suggest a cells lacking WASP. Red panels particularly significant. It is very active in particularly attractive model - that melanoma what steers their direction. Our group aims to improve show F-actin visualised with Group Leader cells generate chemotactic gradients understanding using a wide range of models and techniques. RFP-lifeact and green panels extending pseudopods and very unstable, Robert Insall show Arp2/3 complex. Normal explaining its rarity. We are now seeking the themselves, before migrating up them. Thus cells (top) are well-organised and phosphatases. Unexpectedly, we have also cells drive their own dispersal through polarised away from the site of Research Scientists shown that nearly all the same signals regulate chemotaxis. We have also used chemotaxis Cell movement is a central part of biology the membranes inside and at the leading edge cell division. WASP mutants Xiaoli Du2 the localisation of SCAR and its relative WASP. chambers to show that melanoma cells are involved in embryo development, wound of cells. We are particularly interested in (bottom) are disorganised, with a Jason King3 We are now seeking to understand what those exquisitely chemotactically sensitive. We are 4 healing and immune responses. We are the family of proteins that turns on the pronounced midbody. Jan Ohotski signals are, and how they connect to upstream now working on the molecular details of the Olivia Susanto interested in two related processes. The first is Arp2/3 complex. attractant in serum and the chemotactic chemotaxis, in which external signals orient and signalling molecules such as receptors and receptors that detect it. Scientific Officer attract cells, and which is increasingly seen as a One such regulator is SCAR. SCAR proteins - G-proteins. Peter Thomason fundamental cause of metastasis. The second is also called WAVEs - are important regulators of We are collaborating with the Mathematics the regulation of the Arp2/3 complex, an cell movement. Mutants in a variety of species We have also investigated the roles of another Graduate Students regulator of the Arp2/3 complex, WASH, which Department, University of Strathclyde to make Clelia Amato4 assembly of proteins that promotes movement show that SCAR is required whenever cells need computational models representing moving José Batista1 by driving the formation of actin microfilaments. to make large structures such as lamellipods; is important for cancer cell invasion and plays a cells. Our models already faithfully mimic some Andrew Davidson without it such structures are either small and fundamental role in the sorting of vesicle Yvette Koh4 aspects of the movement of Dictyostelium cells. Most mammalian cells use pseudopods made malformed, or completely absent. SCAR is contents. Gene knockouts in Dictyostelium We are now using the model to test our of polymerised actin to power migration. Our found as part of a five-membered complex with have defects in the intracellular transport of 1 FCT vesicles, specifically in the retrograde sorting of predictions about the underlying mechanisms 2 research focuses on the proteins and pathways the Rac-binding protein PIR121, Nap1, Abi and until December of chemotaxis, and the proteins that are that control these pseudopods. We use three HSPC300. Without the other members of the proteins such as the vacuolar ATPase from 3 until August involved. We are showing that chemotaxis is approaches. For genetic studies we use complex, SCAR is rapidly removed from the cell. lysosomes. This makes mutant cells retain huge 4 from October mostly likely mediated by several dissimilar Dictyostelium, taking advantage of its ease of The prevailing view is that all these proteins act amounts of labelled dextran long after normal mechanisms acting in parallel, including manipulation, and prominent cell movement simultaneously as a huge, homogenous cells have expelled it. We are using this regulated pseudopod growth, pseudopod and chemotaxis. To apply our knowledge to complex that couples Rac signalling to actin observation to drive genetic screens for new retraction and the control of adhesion. Our cancer, we use melanoma cell lines as well as polymerisation. However, our genetic studies WASH interactors. We have also found that theory of self-generated gradients implies that tumours from mouse models and, when in Dictyostelium show that each complex FAM21, a subunit of the WASH regulatory cells behave in a similar way to herds of animals possible, from patients. We also develop member may have a different function – with complex, works differently from the others. in the wild. We are therefore also collaborating computational models of single cells in Nap1 controlling adhesion and Abi inactivation Instead of making actin on vesicles, it couples with mathematical ecologists at the University collaboration with the Mathematics of movement during processes like cytokinesis. the WASH complex to the cell’s cytoskeleton of Glasgow to determine whether this Department, University of Strathclyde, and of These studies point to the SCAR complex and thus allows it to be recycled. We are now comparison can yield useful predictions about populations of moving cells with the Institute of being a nexus integrating multiple inputs - focusing on the mechanism that connects cancer cell behaviour. Biodiversity Animal Health and Comparative signalling, adhesion and a cell’s internal state - FAM21 to the actin cytoskeleton. Medicine, University of Glasgow. In the long and coupling the integrated output to cell Publications listed on page 79 term, we will work on anything that will help us movement. Mechanisms underlying chemotaxis to understand the conserved and fundamental Chemotaxis is emerging as a major driver of mechanisms that drive cell movement. Our experiments are currently focused on tumour metastasis. We have shown that in identifying what regulates each component of Dictyostelium cells it works by a different Regulators of the Arp2/3 complex the complex. SCAR and the other complex mechanism than that which is usually described. Actin drives nearly all cell movement, and the members are phosphorylated at multiple sites Pseudopods are constantly generated in principal driver of actin is the Arp2/3 complex. but the biological significance of these is not random directions, then the ones that point in When turned on, the Arp2/3 complex causes understood. We have shown that control of the best directions are selected and maintained. new actin filaments to form and push against SCAR phosphorylation is important – nearly all

28 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE CELL MIGRATION AND CHEMOTAXIS 29 Figure 2 Sansom). We are interested to know whether the MIGRATION, INVASION AND METASTASIS Expression of fascin in role of N-WASP in invadopodia translates into a well-differentiated differences in tumour formation, progression www.beatson.gla.ac.uk/laura_machesky mouse PDAC. Photo shows immunofluorescence and spread in these models. staining of a murine tumour expressing Pdx-1Cre KRasG12D We are also studying the role of fascin-1 in and p53R172H. Fascin is green, pancreatic ductal adenocarcinoma progression, E-cadherin is white, Slug is a project initiated by former student Ang Li (now red and DNA is blue. Photo by Ang Li. at Rockefeller University in New York) and now continued by Amelie Juin, Hayley Morris and students Emma Woodham and Loic Fort, and in collaboration with Jen Morton and Owen Cancer metastasis requires cells to break away from the Sansom (Fig. 2). We found that fascin is a target Figure 3 of the epithelial to mesenchymal transition in primary tumour and to survive in a variety of environments Hindbrain immunostain showing PECAM for blood pancreatic cancer and has a key role in early before settling into a new site. We aim to gain insights into vessels (green), fascin (red) tumour formation as well as metastatic and DNA (DAPI, blue) and dissemination. Fascin mediates metastasis to the mechanisms of metastatic spread by determining the roles co-localisation in white of fascin peritoneal cavity via the formation of actin-rich with endothelial cells. From Ma filopodia that enable cells to intercalate and of key actin cytoskeletal proteins, such as the actin filament et al., 2013. Photo by Ang Li. migrate through mesothelial cell layers and nucleation machinery and the bundling protein fascin, in seed a new tumour. Fascin also contributes to cancer cell invasive and migratory behaviour. The actin liver metastasis. This work complements our Group Leader cytoskeleton is important not only for cell strength and ongoing efforts to develop fascin inhibitor Laura compounds together with Martin Drysdale migratory capacity but also for adhesion-dependent survival, (Drug Discovery). Machesky membrane trafficking and establishment of polarity. We aim native basement membranes. We speculate that Role of actin regulatory proteins in Research Scientists megakaryocytes may use podosomes for melanoblast migration and melanoma Amelie Juin to understand how various actin regulators control these Ben Tyrrell extending proplatelet arms through the walls of We also started new projects with Emma processes and thus contribute to tumour initiation, growth sinusoids or blood vessels to release platelets Woodham and postdoc Ben Tyrrell on the role Scientific Officer and spread as well as to fundamental mechanisms of into the bloodstream. In our review (Schachtner of Rho family GTPases in melanocyte migration. Heather Spence et al., Cytoskeleton 2013; 70: 572), we discuss We previously showed that loss of Rac1 causes mammalian development. podosomes as mechanosensors and mediators major defects in melanoblast migration and Clinical Research Fellows Hayley Morris1 of adhesion and matrix remodelling and the proliferation during development (Li et al., Dev relationship between podosomes and cancer Cell 2011; 21: 722) and now we are studying the Richard Stevenson Role of actin nucleating proteins in cell invasive structures such as podosomes and cell invadopodia. Hannah has taken up a roles of RhoA and Cdc42 in melanoblasts, migration, invasion and membrane trafficking invadopodia. Graduate Students postdoc position with Tanja Maritzen in together with Cord Brakebusch (University of The Arp2/3 complex is the major inducer of Loic Fort Berlin, while Simon has moved on to a Copenhagen, Denmark). With postdoc Yafeng Emma Woodham actin filaments in response to extracellular Together with former postdoc Xinzi (Amber) Yu lectureship in Hull. Ma, we published this year that loss of fascin signals. Members of the Wiskott-Aldrich and scientific officer Heather Spence and in causes defects in melanoblast migration and Visitor Syndrome Protein family (including WASP/N- collaboration with Lynn McGarry and Emma Emmy Borgmastars We also continued our studies of the role proliferation that are subtler than loss of Rac1 WASP, SCAR/WAVE, WASH, WHAMM and JMY) Shanks (RNAi Screening), we completed a of WASH in actin networks of Dictyostelium (Ma et al., Development 2013; 140: 2203). Fascin transmit signals to the Arp2/3 complex to trigger targeted screen for genes regulating invasion. 1 MRC cells (together with Robert Insall, Park et al., is transiently expressed in murine melanoblasts actin assembly. Each of these proteins is We continue to validate and study several new Dev Cell 2013; 24: 169) and in breast cancer during their migration and is lost later when they regulated differently and one of our aims is to targets involved in three-dimensional migration cells. Postdoc Tobias Zech found a potential differentiate into melanocytes. Fascin is understand the mechanisms of regulation and and invasion. The most promising will be taken connection between WASH and ERBB2 expressed in some melanomas but is not tightly the involvement of these proteins in invasion forward into in vivo screens for invasion and signalling, which we are studying further in correlated with stage or progression as it is in and metastasis of cancer as well as their normal metastasis. Amber has moved on to a vitro and in vivo with the help of a pilot grant many epithelial cancers. cellular function. WASP family proteins regulate postdoctoral position with Iain McNeish from Breast Cancer Campaign. Tobias has actin assembly in multiple essential and (University of Glasgow). recently taken up a lectureship at the Fascin is also present in tumour stroma and is pathological cellular processes, such as University of Liverpool. expressed by vascular endothelial cells, smooth endocytic trafficking, protrusion of lamellipodia This year, we (student Hannah Schachtner and muscle cells and pericytes. Despite the and filopodia, cell division and assembly of postdoc Simon Calaminus) published a study Role of actin regulatory proteins in abundance of fascin in blood vessel walls, we showing that the platelet precursor cells, Figure 1 colorectal and pancreatic cancer found that transplanted B16 melanomas megakaryocytes assemble actin-based Mouse megakaryocyte labelled Clinical research fellow Richard Stevenson showed only slight differences in vascularisation podosomes that resemble invadopodia of with phalloidin (actin filaments, identified a role of the actin bundling protein when the host was a fascin knockout. Tumour red), ARPC2 antibody (Arp2/3 cancer cells (Fig. 1 and Schachtner et al., Blood fascin in tumour formation in colorectal cancer growth was not affected by these small complex, green) and DAPI (DNA, 2013; 121: 2542). Megakaryocyte podosomes models as well as a potential role in colitis. We differences, making fascin dispensable for blue). The prominent dots are contain actin, Arp2/3 complex and WASP and podosomes. Photo by Hannah started new projects with MRC funded clinical tumour angiogenesis. Likewise, developmental they are used for adhesion, spreading and Schachtner. research fellow Hayley Morris on the role of angiogenesis does not seem to be greatly motility. Podosomes form an interconnected N-WASP in colorectal cancer (in collaboration affected by loss of fascin (Fig. 3 and Maet al., network in cells that can sense and respond to with Owen Sansom) and with postdoc Amelie Biol Open 2013; 2: 1187). changes in the extracellular matrix environment. Juin on the role of N-WASP in pancreatic ductal We also found that megakaryocyte podosomes adenocarcinoma (with Jen Morton and Owen Publications listed on page 81 could degrade matrix and protrude through

30 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE MIGRATION, INVASION AND METASTASIS 31 INTEGRIN CELL BIOLOGY www.beatson.gla.ac.uk/jim_norman

Integrins are cell surface receptors that engage the extracellular Figure 1 Influence of RCP-drivenα 5β1 matrix and help cells to move. Cancer cells use integrins in recycling on localisation of RacGAP1 and downstream order to migrate away from primary tumours to form signalling to RhoGTPases. (A) In p53 null cells, p63 metastases. Like many other receptors, integrins are suppresses association of RCP with α5β1, so the integrin is not internalised (or endocytosed) from the cell surface into trafficked to the cell front and Rac signalling predominates at endosomes. Once within endosomes, integrins can either be the leading edge. (B) Gain-of- sorted for degradation or can recycle to the cell surface via a function mutant p53 inhibits p63 Group Leader to promote association of RCP Figure 1 number of different routes. We have found that a range of with α5β1. Production of Jim Norman phosphatidic acid by DGKα drivers of cancer metastasis operate by altering the way in within the tips of protruding Research Scientists pseudopods recruits RCP/α5β1/ Joanna Birch which integrins recycle to the plasma membrane. Moreover, EGFR1 vesicles, and localises Jesica Diaz-Vera1 we now know that components of the integrin recycling downstream signalling via Akt to Emmanuel Dornier this subcellular region. Here Akt Elena Rainero2 machinery influence clinical outcomes in patients with phosphorylates RacGAP1, Peter van den Berghe allowing inactivation of Rac and pancreatic and breast cancer. We will continue to assemble a activation of RhoA to promote Associate Clinical Scientist pseudopod extension and Iain MacPherson detailed molecular picture of integrin recycling and how this invasion.

Scientific Officer contributes to metastasis, and we hope to reveal which are the Figure 2 Louise Mitchell Schematic diagram depicting most promising components of the pathway to target for the relationship between Graduate Students cancer therapy. trafficking ofα 5β1 and mTOR Cassie Clark activation at late endosomes/ Figure 2 Christine Gundry lysosomes. Nikki Heath Elena Ruengeler Use of phosphoproteomics to characterise extracellular matrix, in a RhoA-dependent lysosomes and their degradation within these compare the distribution of proteins between RhoGTPase signalling downstream of mutant manner. compartments. Integrin internalisation from the plasma membrane and recycling 1 Fundacion Canaria Doctor p53/RCP-dependent trafficking fibrillar adhesions is required for proper endosomes under control conditions and when Manuel Morales We knew from our previous work that Rab- An endocytic pathway transporting ligand- lysosome positioning, and recruitment and RAB17 levels are suppressed. By careful analysis 2 West of Scotland Women’s coupling protein (RCP) dependent recycling occupied integrins from fibrillar adhesions to activation of mTOR at this cellular sub- of peptides from proteins that are differentially Bowling Association influences the way thatα 5β1 integrin signals to late endosomes supports lysosomal compartment. These findings identify integrin trafficked, we have identified VAMP8 as a novel Rho subfamily GTPases. As RhoA signalling is recruitment and activation of mTOR endocytosis as a mechanism to support mTOR effector of RAB17 and have gone on to show likely important to dictating how RCP controls Recycling of internalised integrin is known to signalling and point to the extracellular matrix as that RAB17/VAMP8 influences invasion by invasion, we used a phosphoproteomic occur at the front and back of migrating cells to a possible source of nutrients for invading controlling trafficking of neuropilin 2 (NRP2). approach to look at signalling events coordinate membrane protrusion and retraction cancer cells (Fig. 2). Thus, we have developed a new mass downstream of RCP trafficking. From this, we respectively but less is known about the spatio- spectrometry approach that is capable of have identified a novel PKB/Akt substrate, temporal organisation of integrin endocytosis Use of SILAC mass spectrometry to identify identifying novel effectors and cargoes of Rab RacGAP1, which is phosphorylated as a and the processes that this controls. We have new components of the invasive ‘recyclome’ GTPases that function during cancer invasion. consequence of RCP-dependent α5β1 developed a novel photoactivation-in-TIRF We have used gene expression arrays to identify We anticipate that further characterisation of the trafficking. Phosphorylation of RacGAP1 approach to show that ligand-bound integrins RAB17 as a gene whose expression must be invasive ‘recyclome’ will identify more new promotes its recruitment to IQGAP1 at the tips are internalised at fibrillar adhesions (but not at suppressed in order for MAP kinase signalling receptor cargoes that are key to invasion, and of invasive pseudopods. Live cell imaging focal adhesions) located under the nucleus and pathways to drive invasive migration. To identify that these may represent new targets at which experiments using FRET reporter probes for Rho are trafficked directly to nearby late endosomes/ which of the receptor cargoes of RAB17 are to aim anti-cancer drugs. subfamily GTPases showed that phospho- lysosomes without passage through early responsible for its ability to oppose tumour cell RacGAP1 locally suppresses the activity of Rac endosomes. This clathrin-independent invasion, we have developed a novel SILAC Publications listed on page 82 and promotes the activity of RhoA in this internalisation route requires tensins-1, -2 and -3 (stable isotope labelling in cell culture) subcellular region (Fig. 1). This Rac to RhoA and the class II Arf subfamily GTPase, Arf4. approach. By using this quantitative mass switch promotes extension of pseudopodial Suppression of tensins or Arf4 disrupts flow of spectrometry approach in combination with processes and invasive migration into the ligand-bound integrins to late endosomes/ membrane purification methods, we are able to

32 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE INTEGRIN CELL BIOLOGY 33 MOLECULAR CELL BIOLOGY www.beatson.gla.ac.uk/michael_olson

A major function of the actin cytoskeleton is to provide the of reactive oxygen species in cell protrusions and dynamic changes in the actin cytoskeleton structural underpinning that gives a cell shape and mechanical that take place in this region to enable cell strength. The actin cytoskeleton is dynamic, undergoing migration.

constant rearrangement and reorganisation in response to LIM kinases in prostate cancer external factors. Alterations to the cytoskeletal architecture Prostate cancer affects a large proportion of the male population and is primarily driven by have significant consequences on the entire cell - such as androgen receptor (AR) activity. First-line morphology, cytokinesis, adhesion and motility - but also at treatment typically consists of reducing AR signalling by hormone depletion but resistance Group Leader the subcellular level. As well as these structural functions, the Michael Olson inevitably develops over time. One way to actin cytoskeleton additionally acts as a scaffold, bringing overcome this issue is to block AR function via Research Scientists alternative means, preferably by inhibiting Mads Gabrielsen1 together proteins that not only contribute to cell shape but also protein targets that are more active in tumours Felipe Lourenco proteins that have diverse activities such as signal transduction than in normal tissue. By staining prostate Katerina Mardilovich1 cancer tumour sections, elevated LIM kinase 1 Nicola Rath and gene transcription. The scaffolding function is not limited (LIMK1) expression and increased Mathieu Unbekandt2 phosphorylation of its substrate cofilin were Serge Zander to the spatial organisation of protein complexes as the actin found to be associated with poor outcome and Scientific Officer cytoskeleton may also recruit or stabilise specialised reduced survival in patients with non-metastatic June Munro membrane domains. prostate cancer. Pharmacological inhibition of LIMK activity reduced proliferation and Clinical Research Fellow Jennifer Brown3 increased apoptosis in androgen-dependent Crystal structure of human cofilin 1 crystallography to elucidate how it could Figure 1 role is the actin binding protein, cofilin 1. Cofilin prostate cancer cells more effectively than in Graduate Students The actin cytoskeleton is the chassis that gives a interact with and regulate dynamic actin Hydrogen peroxide is generated 1 has been demonstrated to increase the rate of androgen-independent prostate cancer cells. Jenifer Cameron4 in protrusions during cell cell its shape and structure, and supplies the cytoskeletal structures. Although wild-type actin monomer (G-actin) dissociation from the LIMK inhibition blocked ligand-induced AR Linda Julian movement. Fluorescence nuclear translocation, and reduced AR protein Dominika Rudzka power for numerous dynamic processes human cofilin 1 proved to be recalcitrant, a lifetime imaging was used to pointed end of filamentous actin (F-actin) and to including motility, endocytosis, intracellular C147A point mutant yielded crystals that detect changes in the optical sever F-actin. Additionally, at high stability and transcriptional activity, consistent Visiting Students transport and division. To perform these diffracted to 2.8 Å resolution. These studies properties of the hydrogen concentrations cofilin can promote the with its effects on proliferation and survival Laura Bello Figueroa activities, the cytoskeleton undergoes constant revealed how the actin binding helix undergoes peroxide biosensor probe nucleation of actin filaments and actin rod acting via inhibition of AR activity. Furthermore, Dominika Kowalczyk HyPer-cyto. Subcellular changes remodelling and reorganisation. One of the a conformational change that increases the formation. A further post-translational inhibition of LIMK activity increased αTubulin Haneela Qadir in fluorescence lifetime are acetylation and decreased AR interactions with Maike Schuldt major actin remodelling families is the cofilin number of potential hydrogen bonds available indicated by the heat map, with modification that can alter cofilin function is Tubulin, indicating that the function of LIMK in Stephanie Schumacher family of proteins, made up of cofilin 1, cofilin 2 for substrate binding. magnitudes of these changes cysteine oxidation, which predominantly results α and actin depolymerising factor (ADF), that indicated in nanoseconds (ns) on in the establishment of disulphide bonds. regulating microtubule dynamics contributes to 1 CRUK Discovery sever aged ADP-associated actin filaments to Regulation of cofilin 1 activity by cysteine the left. Boxed regions show AR function. Finally, treatment of androgen- higher magnification of Committee reduce filament length and provide new oxidation We found that H O levels are elevated in independent prostate cancer cell lines reduced hydrogen peroxide being 2 2 2 MRC DPFS cell motility, indicating that LIMK inhibitors could potential nucleation sites. Despite the significant Cell migration is essential for many processes produced in cell protrusions, migrating relative to stationary cells with the 3 CRUK Glasgow Centre interest in cofilin as a central node in actin including embryonic development, wound relative to lower hydrogen highest levels being observed at cell protrusions. be beneficial for the treatment of prostate 4 until October cytoskeleton dynamics, to date the only forms healing and inflammation. It is also involved in peroxide in the cell body. In addition, protein oxidation is increased in cancer both by reducing nuclear AR of cofilin for which crystal structures have been the local invasion and metastasis of cancer cells. migrating compared to stationary cells and we translocation, leading to reduced proliferation solved are from the yeast, Chromalveolata and In order for cells to migrate the actin-myosin identified cofilin as one of these oxidised and survival, and by inhibiting prostate cancer plant kingdoms; none have previously been cytoskeleton must be constantly rearranged, proteins. Moreover, the oxidation of cofilin cell dissemination. reported for an animal cofilin protein. Two which is mediated by a number of proteins that inhibits its ability to decrease F-actin levels and distinct regions in animal cofilin are significantly act downstream of the Rho GTPases. These this is dependent on cysteine residues 139 and Publications listed on page 83 larger than in the forms previously crystallised, proteins are tightly regulated both spatially and 147. The oxidation of these cysteine residues is suggesting that they would be uniquely temporally to ensure that cell migration occurs also required for cell adhesion and directional organised. Therefore, we sought to determine at the appropriate time and in the correct migration. Taken together these results provide the structure of human cofilin 1 by X-ray direction. One of the proteins that has a central a direct link between the increased production

34 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE MOLECULAR CELL BIOLOGY 35 COLORECTAL CANCER AND WNT SIGNALLING www.beatson.gla.ac.uk/owen_sansom

Colorectal cancer is the third most common cancer in the UK 7 MRC In colorectal cancer, KRAS is often co-mutated Mechanistically, Kras mutation causes the 8 Wellcome Trust with APC (in approximately 40% of cases). These activation of mTORC1 targets in an mTORC1- and the second most common cause of cancer mortality. The 9 joint with A*STAR mutations are activating and tumours carrying independent manner. We are now examining focus of our group is to understand the early changes 10 CRUK Glasgow Centre KRAS mutations have a poorer prognosis and the pathways of resistance in these cells and 11 are less likely to respond to therapy. Therefore, hope to come up with rationale combination associated with intestinal neoplasia in order to identify novel until October 12 until November my laboratory has been investigating the therapies that will overcome this resistance cooperation of Apc and Kras mutations in mechanism and specifically target cells carrying markers of the disease as well as new targets for therapy. The 13 from November models of colorectal cancer. The additional both of these mutations. Importantly, loss of 14 until August key intestinal tumour suppressor is the APC gene that is mutation of Kras exacerbates many of the RAC1 or mTORC2, a less well studied member 15 from October phenotypes of Apc loss, increasing proliferation of the mTORC family, both appear to be mutated in approximately 80 percent of sporadic cancers. 16 until February rates and accelerating tumourigenesis. required for the proliferation of cells carrying a Group Leader Central to our work is the use of novel inducible models of Owen Sansom Importantly, APC deficient cells that also carry a KRAS mutation in a number of different cancers. intestinal tumourigenesis that allow us to study the functions of Kras mutation are intrinsically resistant to FRSE mTORC1 inhibition. This may help explain why Publications listed on page 84 specific tumour suppressor genes. later stage colorectal cancer trials using Research Scientists mTORC1 inhibitors have not worked. Patrizia Cammareri1 Fatih Ceteci1 Rac1 integrates ROS and NF- B signalling to inhibitors of Rac1 are available, our data suggest Julia Cordero2, 11 κ drive hyperproliferation, stemness and that these could be of benefit in colorectal Figure 1 William Faller3 In vivo multiphoton imaging of Dritan Liko4, 12 transformation following Apc loss cancer. the intestinal stem cell marker Bryan Miller We have previously shown that MYC is the key Lgr5 shows Rac deficiency Claudio Murgia effector downstream ofApc loss in vivo. Since it mTORC1 is required for the proliferation of suppresses the increased levels Kevin Myant5 may be difficult to pharmacologically inhibit APC deficient cells followingApc loss, following Apc loss. In Alessandro Scopelitti6, 11 transcription factors such as MYC, investigating although this requirement is lost following collaboration with Kurt Myriam Solar Abboud Anderson, we have quantified Ee Hong Tan more druggable targets of the Wnt/c-Myc additional Kras mutations the levels of the stem marker David Vincent3 pathway within the intestine may reveal Another pathway that is activated following Apc potential therapeutic targets for colorectal loss, in a MYC-dependent manner, is the Lgr5 through the use of a reporter GFP in murine intestinal Scientific Officers cancer. Recent work in the laboratory has mTORC1 signalling pathway. mTORC1 is a kinase whole-mounts. As can be seen Tam Jamieson shown that Rac GEFs and the active Rac1 splice that is required to coordinate proliferation and here, there is a marked increase Rachel Ridgway in the amount of Lgr5-GFP Andrew Campbell3, 13 isoform, Rac1b are upregulated following Apc cell growth, and has long been viewed as an loss and Rac1 is required for the attractive pathway for cancer therapy. The following Apc loss in vivo (APC), however following combined Clinical Research Fellows hyperproliferative and progenitor/stem cell major reason for this is that there are a number loss of Apc and Rac (APC RAC) Douglas Morran7, 14 phenotypes that ensue (Fig. 1). Mechanistically, of inhibitors of this pathway that work well in this is restored to wild-type Colin Steele8 Rac1 is required for the production of ROS vivo and are either licensed for use in patients or levels. Importantly, loss of Rac alone (RAC) has equivalent levels Graduate Students following Apc loss, which subsequently clinical trials. We have shown that APC deficient of GFP to wild-types. Red here is Sahra Derkits activates NF- B signalling, resulting in p65 cells are absolutely dependent on mTORC1 to κ second harmonics imaging of David Huels1 binding to the promoters of intestinal stem cell proliferate. Genetic inhibition or use of collagen. David Gay3, 15 genes. Physiologically, Rac1 deletion stops rapamycin, a chemical inhibitor, completely Pedro Salgueiro9 adenoma formation following Apc loss and removes the proliferative capacity of APC Figure 2 Inhibition of mTORC stops the MRes Student instead results in small lesions that do not form deficient cells. This is associated with a slowing proliferation of APC deficient Josh Leach10, 16 tumours and lack stem cell markers such as Lgr5 of translational elongation and hence, given the and which recapitulate those formed when Apc massive burst of transcriptionally activity of APC cells. A) Schematic of mechanism of how loss of Apc 1 EU PRIMES is deleted from non-stem cell compartments. deficient cells, cells enter growth arrest (Fig.2). causes the activation of mTORC 2 Royal Society Taken together, our data highlight that Rac1 Although this growth arrest is reversible, as signalling. B) H&E stained 3 ERC activation downstream of Wnt signalling and rapamycin is a well-tolerated drug it is possible sections of intestines, wild-type 4 EMBO upstream of ROS/NF- B signalling is a critical that this would be beneficial for those patients (WT) or APC deficient (APC). As κ can be seen by the red bar, 5 AICR event at the early stages of intestinal that are predisposed to colorectal cancer. wild-type intestinal crypts are 6 NC3Rs (joint with carcinogenesis to maintain the progenitor cell much smaller than APC deficient Marcos Vidal) phenotype. Given a number of preclinical ones. Treatment with rapamycin returns APC deficient crypts (APC + rapamycin) to the same size as wild-type ones.

36 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE COLORECTAL CANCER AND WNT SIGNALLING 37 DROSOPHILA APPROACHES TO CANCER www.beatson.gla.ac.uk/marcos_vidal

We utilise the fruit fly Drosophila to understand fundamental constrains ISC proliferation. Midgut unknown roles of neuroendocrine cells and the neuroendocrine cells secrete Bursicon, which conserved ligand/receptor Bursicon/DLGR2. aspects of cancer biology. We have previously reported an acts as a paracrine factor on the VM through Indeed, while the current knowledge indicates inflammatory reaction by the innate immune system when DLGR2, the ortholog of mammalian LGRs. that LGRs drive stem cell proliferation via Bursicon/DLGR2 restricts ISC proliferation and activation of Wnt signalling, clinical data tumours arise, comparable to the one observed in cancer upregulation of cAMP in the visceral muscle. In indicates they are silenced or mutated in patients. Importantly, powerful fly genetic tools allowed us to the absence of this pathway, the intestine numerous tumours, indicating they can act as hyperproliferates resulting in excess cells and tumour suppressor genes. Our results provide a define for the first time the contexts dictating ‘good’ versus epithelial multi-layering (Fig. 2). Our results mechanistic framework to understand these ‘bad’ inflammation. Recently, we have dissected systemic identify a novel paradigm in the regulation of recent clinical observations. ISC quiescence through modifications of stem Group Leader crosstalk between tumours and peripheral energy stores such Marcos Vidal cell niche signalling, which uncovers previously Publications listed on page 86 as adipose tissue. Remarkably, our data demonstrate that Research Scientists Figure 1 Federica Parisi adipocytes can sense tumours at a distance, and react by Confocal images from Alessandro Scopelitti Drosophila imaginal disc tissues activating pro-inflammatory pathways that act as tissue non- in a transgenic background Scientific Officer autonomous tumour suppressor factors. These results could expressing a Venus-tagged form Yachuan Yu of Eiger/TNF specifically in the shed light on the complex systemic relationships between macrophages (white arrows). Graduate Students Note the vesicles of Christin Bauer tumours, chronic inflammation and peripheral tissues in macrophage-derived TNF Joseph Hodgson incorporated into epithelial Juan Macagno human cancer patients. We also characterised a novel role of tumour cells (yellow arrows), a Rhoda Stefanatos LGR receptors - known as stem cell markers in numerous process absent in normal tissue. Intern tissues - as tumour suppressors directing stem cell quiescence. Paula Climent-Canto

Systemic crosstalk between Drosophila demonstrated that Eiger protein produced by adipocytes and epithelial tumours macrophages is internalised by tumour cells High tumour burden is associated with but not by normal ones (Fig. 1). Altogether, increased levels of circulating inflammatory our results provide a novel paradigm for a cytokines that influence the pathophysiology of long-range tumour suppression function of the tumour and its environment. The cellular adipocytes in Drosophila that may represent and molecular events mediating the organismal an evolutionary conserved mechanism in the response to a growing tumour are poorly organismal response to solid tumours. understood. We revealed a bidirectional crosstalk in Drosophila between epithelial Mechanism for LGRs as tumour tumours and the fat body - a peripheral immune suppressor genes Figure 2 tissue akin the mammalian liver and white The mechanisms regulating stem cell niche Haematoxylin and eosin staining adipose tissue. Epithelial tumours trigger a plasticity remain poorly understood. of paraffin embedded sections systemic immune response through local Mammalian leucine-rich repeat-containing G from wild-type or burs mutant activation of Eiger/TNF signalling that leads to protein-coupled receptors (LGRs) have recently Drosophila intestines. Note the hypercellularity and epithelial the recruitment and activation of hemocytes (fly been characterised as somatic stem cell multi-layering in burs mutant macrophages) and to Toll pathway upregulation markers in multiple epithelial tissues. However, midguts. in distant adipocytes. Reciprocally, Toll elicits a their biological roles in adult tissues are largely tissue non-autonomous programme in unexplored. We have identified a novel function adipocytes that drives tumour cell death. of LGRs as inhibitors of intestinal stem cell (ISC) Hemocytes play a critical role in this system by proliferation in Drosophila. Our results uncover producing the ligands Spätzle and Eiger, which a new paracrine signalling mechanism are required for Toll activation in the fat body composed of neuroendocrine cells and and for tumour cell death. Indeed, we mesenchyme/visceral muscle (VM) that

38 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE DROSOPHILA APPROACHES TO CANCER 39 VASCULAR PROTEOMICS www.beatson.gla.ac.uk/sara_zanivan

Tumour angiogenesis, the process of vessel growth from Figure 1 fibroblasts and immune cells, has a different Quantitative proteomics composition and structure compared to normal in angiogenesis. (a) Schematic pre-existing ones, is a well-recognised hallmark of cancer. tissue. This contributes to tumour progression overview of the formation of a Indeed, tumour vessels provide the oxygen and nutrients new tumour blood vessel from a to malignancy. Accordingly, our in vivo pre-existing one induced by the quantitative proteomic study of mouse skin necessary to sustain the uncontrolled proliferation of tumour secretion of pro-angiogenic carcinogenesis revealed that the ECM factors from tumour cells and composition differs between benign and cells and provide a way for them to escape the primary tumour cancer-associate fibroblasts malignant tumours, and that specific cell (CAFs). The arrows indicate the and form distant metastases. Additionally, in a clinical context, direction of the secreted factors. adhesion proteins are highly expressed in tumour vessels can impact on the success of conventional (b) Schematic overview of a mass tumour cells associated with malignancy Group Leader spectrometry shotgun (Zanivan et al., Cell Reports 2013; 3: 552). In the therapies. Indeed, tumour vessels are generally leaky and proteomic approach used to context of angiogenesis, tumour stroma cells identify pro-angiogenic factors Sara Zanivan such as cancer-associated fibroblasts (CAFs), function poorly, which can result in reduced efficiency of secreted by CAFs. Immortalised Research Scientists normal (iNFs) and cancer- can affect endothelial cell functions by altering Juan Ramon Hernandez chemotherapeutic drug delivery and limited efficacy of associated (iCAFs) human the ECM’s composition and mechanical Francesca Patella radiotherapy. For this reason, much effort is devoted to fibroblasts are used in properties, and by secreting pro-angiogenic combination with SILAC for factors (Fig. 1a). Therefore, there is a clear link quantitative proteomic analysis. Scientific Officer developing therapies that interfere with the angiogenic process between CAFs and the angiogenic process in Lisa Neilson In a typical workflow, SILAC in cancer for use in conjunction with conventional chemo- proteins secreted into the cancer, and we aim to investigate this further. Graduate Students conditioned medium by iNFs Fernanda Kugeratski and radiotherapy. and iCAFs are mixed together, We have applied SILAC-based quantitative Steven Reid fractionated, digested and proteomics to characterise in depth the peptides are analysed by phosphoproteome and proteome of nano-liquid chromatography Visitor Our group is working with state-of-the-art mass When endothelial cells are plated on an Maartje van den Biggelaar (nLC)-high resolution tandem immortalised human fibroblasts of different spectrometry in combination with SILAC (stable appropriate matrix, such as matrigel, they mass spectrometry (MS/MS). MS origins, normal (iNF) and cancer-associated isotope labelling with amino acids in cell culture) assemble into tubular structures complete with data are analysed with the (iCAF), to provide a detailed picture of proteins to perform quantitative proteomic and post- a lumen, and thereby morphologically MaxQuant computational specifically expressed or with an altered level of platform to identify proteins translational modification - such as recapitulate the in vivo process. By using expression in the tumour environment. Since secreted in high levels by iCAFs. phosphorylation - analyses. We combine the SILAC-based quantitative mass spectrometry we observed that iCAFs were more pro- use of this technology with a variety of cellular we have profiled the proteomic changes angiogenic than iNFs in in vitro models of and molecular approaches with the aim of associated with this process and identified the angiogenesis, we have additionally analysed the better understanding the complexity of the proteins CLEC14A and MMRN2 as ECM ECM and conditioned medium produced by signalling that drives (tumour) vessel formation, components required for angiogenesis in vitro, these cells by mass spectrometry with the aim with a particular focus on how the environment and blood vessel markers modulated during of identifying previously uncharacterised surrounding newly forming vessels impacts on tumour progression in genetic mouse models particular focus on lipid metabolism. We aim pro-angiogenic factors (Fig. 1b). We are this process. of multistep carcinogenesis (Zanivan et al., Mol to integrate these data with those previously currently investigating the functions of some Cell Proteomics 2013; 12: 3599). Additionally, generated to draw a more comprehensive of the identified proteins in the context of Endothelial cell signalling in angiogenesis this proteomic study highlighted that the picture of the molecular mechanisms angiogenesis using in vitro, ex vivo and in Endothelial cells constitute the first layer of the morphogenetic process is associated with regulating the formation of new vessels, and vivo models. vessel wall and are the major cell players in the major metabolic changes in endothelial cells, to identify and further characterise new key angiogenic process. Indeed, endothelial cells and that this alteration is functional. We are proteins as possible targets to interfere with In addition, we are exploring the signalling orchestrate a multitude of cellular mechanisms currently investigating this aspect further during tumour vascularisation in the context networks activated in endothelial cells by the that trigger the formation of new and mature using a unique combination of proteomics, of anti-cancer therapies. ECM’s mechanical properties and composition, vessels – for example, they sprout, digest and mathematical modelling (in collaboration which are typically altered in the tumour migrate through the extracellular matrix (ECM), with Eytan Ruppin, University of Tel Aviv) The extracellular environment drives tumour environment. This will hopefully reveal signalling proliferate, change morphology and recruit and metabolomics (in collaboration with progression and angiogenesis and identify specific proteins involved in tumour mural cells. A better understanding of the Eyal Gottlieb). The environment surrounding cells strongly angiogenesis. Candidate proteins will be further molecular mechanisms underpinning affects their behaviour. Importantly, the characterised as potential suitable targets angiogenesis are required in order to be able to Our future work will be devoted to better extracellular environment is diverse in to interfere with the process of angiogenesis interfere more efficiently with this mechanism in characterisation of the complex signalling physiological and pathological conditions. In in cancer. pathological conditions such as cancer. network during vessel formation with a cancer, the tumour stroma, composed of ECM and non-cancer cells such as endothelial cells, Publications listed on page 87

40 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE VASCULAR PROTEOMICS 41 DRUG DISCOVERY

CANCER RESEARCH UK BEATSON INSTITUTE

Martin Drysdale - Drug Discovery Programme

42 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE DRUG DISCOVERY PROGRAMME 43 DRUG DISCOVERY PROGRAMME www.beatson.gla.ac.uk/ddp

The vast majority of deaths from cancer result directly from metastatic spread of the disease, however there are currently no therapeutics specifically designed to combat this process. During the past year we have made significant advances in two of our projects targeting novel approaches to inhibiting invasion and metastasis; these are the kinase MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase), a key effector Group Leader Figure 2 selective cell-based assays where we Elevated fascin expression is strongly correlated in the Rho signalling pathway, and the actin-bundling protein Mice bearing subcutaneous specifically target MRCK (IC50 = 0.05mM) with invasiveness and poor clinical outcome in Martin MDA-MB-231 D3H2LN tumours β fascin. We have also continued to utilise our fragment-based or ROCK1 (IC > 30mM) (Fig. 1). a variety of tumour types, therefore making Drysdale were treated with BDP- 50 hit identification expertise to target other protein-protein 00007215 (10mg/kg, i.p.) and fascin a compelling drug discovery target. We tumour sections stained for Using BDP-00007215 we have carried out have now solved multiple crystal structures of Chemistry expression of phospho-Ser990 Catherine Barber interactions that, although challenging biological targets, have pharmacokinetic/phamacodynamic (PK/PD) in fragments bound to fascin and these have using a phospho-specific Simone Belshaw vivo studies in mice bearing MDA-MB-231 been shown to bind to four distinct sites within a high degree of validation as cancer targets. In particular, we antibody developed in the Olson Justin Bower D3H2LN subcutaneous breast tumours. At the four -trefoil domains that make up the lab. (A) Immunohistochemistry β Keneth Davies are targeting the RAS oncogene, which is one of the best carried out at 15, 30 and 60 10mg/kg i.p. administration BDP-00007215 fascin structure (Fig. 3). Emma Dick minutes post dose. (B) Semi- reduces the staining of active MRCK using the Stuart Francis α validated cancer targets and found to be mutated in 30% of all quantitation of staining using the Kate Gill phospho-specific antibody developed in Site-directed mutagenesis data derived weighted Histoscore method. Duncan McArthur human cancers. collaboration with the Olson lab (Fig. 2). in-house and also from published literature Charles Parry Additional analogues are being profiled inin has directed us to two of these sites as being Angelo Pugliese vivo pharmacodynamic (PD) studies ahead of most likely to be functionally relevant and Mairi Sime Myotonic dystrophy kinase-related Cdc42- selection of the best compound for in vivo these have been prioritised for further study. binding kinase (MRCK) Biology efficacy studies. Using structure-guided drug design we Mounting evidence suggests MRCK as an Diane Crighton have developed hits for these two sites. Dan Croft attractive target for anti-metastatic therapy. Fascin Improvements in affinity of 10-1000 fold Laura McDonald MRCK is a key effector in the Rho GTPase The actin-binding protein fascin crosslinks have been achieved and we now have >60 Patricia McConnell signalling pathway that acts to promote cell filamentous actin into tightly packed bundles crystal structures of ligands bound to our Mokdad Mezna movement by initiating actomyosin contractility that drive the formation of cell surface two main sites of interest. and reorganisation of the actin cytoskeleton. An Structural Biology protrusions involved in cell migration and Kenneth Cameron increasing number of tumour types show degradative invasion into extracellular matrix. Publications listed on page 77 Gillian Goodwin mis-regulated expression of a number of Chris Gray components of this pathway. Elevated Rho Figure 3 Jen Konczal GTPase expression, loss of negative regulators Fragments identified binding to Andrew Pannifer and/or upregulation of downstream effectors four distinct sites on fascin Alex Schuettelkopf such as MRCK, results in enhanced signalling through the Rho GTPase pathway (Unbekandt and Olson, J Mol Med 2014; 92: 217). Figure 1 MDA-MB-231 D3H2LN cells As part of a collaboration with Mike Olson’s stably expressing tetracycline group, we are developing small molecule inducible MRCKβ (top panel) or ROCK1 (bottom panel) were inhibitors that target the α and β isoforms of treated with BDP-00007215 as MRCK. These inhibitors could be used either indicated in the presence of alone or in combination for the management of doxycycline. Levels of metastatic disease. Our lead series has delivered phosphorylated MLC (pMLC; BDP-00007215, which has high potency for lower band) and α-Tubulin (upper band) were measured by MRCKα and β and excellent selectivity over western blotting and quantified other key kinases such as Rho kinases (ROCKs). by LI-COR infrared scanning. This translates into exquisite selectivity in our

44 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE DRUG DISCOVERY PROGRAMME 45 ADVANCED TECHNOLOGIES

CANCER RESEARCH UK BEATSON INSTITUTE

Kurt Anderson - Beatson Advanced Imaging Resource (BAIR) Gabriela Kalna - Bioinformatics and Computational Biology Gillian Mackay - Metabolomics Nick Morrice - Proteomics and Mass Spectrometry Emma Shanks - RNAi Screening Karen Blyth - Transgenic Models of Cancer Douglas Strathdee - Transgenic Technology

46 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 47 BEATSON ADVANCED IMAGING RESOURCE (BAIR) BIOINFORMATICS AND COMPUTATIONAL www.beatson.gla.ac.uk/advanced_technologies BIOLOGY www.beatson.gla.ac.uk/advanced_technologies

Light microscopy is a fundamental technique in cell biology The unit provides support for a range of research projects that and cancer research. The development of genetically encoded require computational approaches, advanced statistical fluorophores (fluorescent proteins) has revolutionised research analyses or mathematical modelling. Despite the continuing by enabling direct visualisation of any gene product. demand for data analysis from high-throughput technologies, we strive to ensure that even the smallest task receives our full Concomitant with the development of new between commercial partners and users. We attention in terms of experimental design, the application of genetic tools, there have been tremendous also provide vision for future trends and help to advances in fluorescence imaging technology identify new technology of potential benefit to appropriate statistical tests and the clear presentation of results Head to visualise molecular dynamics in living cells, our researchers. Head for use in publications. Kurt Anderson tissues, and organisms. These powerful Gabriela Kalna techniques are increasingly sought by Changes this year Staff Scientist researchers, who require assistance in both the This year we celebrated our first published use Informaticians Our team focuses on exploratory data analysis, chemotaxis, such that simulated cells are Gaurav Malviya evaluation and application of imaging of PET with one of Kevin Ryan’s papers: Ann Hedley with the ultimate goal of providing insights that capable of interacting with their environment. Matthew Neilson enhance our understanding of cancer biology. This exciting development provides us with the Scientific Officers technology in order to address fundamental (Rosenfeldt et al., Nature 2013; 504: 296). In Tom Gilbey questions in cancer biology. order to meet the rising demand for preclinical We offer routine processing of data, differential computational capability to simulate a wide Ewan McGhee use of PET, SPECT and CT, Gaurav Malviya was expression, supervised and unsupervised range of processes, such as phagocytosis and Agata Mrowinska Our mission is to support basic imaging and the recruited to a Staff Scientist post. Gaurav brings machine learning, and graph and network contact inhibition. We will also employ this Margaret O’Prey development of advanced applications. Basic expertise in radiolabelling and characterisation theory-based analyses. Many of our data model to investigate the effect of membrane David Strachan imaging support primarily consists of training of antibodies for SPECT and PET imaging to the analysis tasks are performed using specialised rigidity on a cell’s ability to migrate through users in simple acquisition and analysis Beatson, and will lead on tracer development computer software such as Partek Genomics constrictions of varying widths. techniques. Development of advanced projects. In May we began the installation of a Suite, MATLAB (most notably the Bioinformatics applications requires close work with our users new multiphoton TRIM microscope from and Statistics Toolboxes) and the open source Finally, we have been developing a particle- to understand their scientific questions and help LaVision Biotec. This system will have increased Bioconductor package for R. We also make use based spatial simulator that aims to provide them develop appropriate imaging strategies. sensitivity through the use of hybrid detectors of analytical routines that have been developed some quantitative insight into the role of The following advanced techniques have been for both intensity and lifetime imaging. It will in-house in collaboration with our colleagues in E-cadherin interactions in cell-cell adhesions. identified as important: medium throughput also have a ‘cloud scanner’ that increases signal Mathematics, Statistics, Computer Science and E-cadherin molecules are capable of forming long term time lapse imaging; high resolution and minimises photo-damage by scanning the Biology. In addition to Ingenuity Pathway three distinct interaction types, which allow live cell imaging; confocal microscopy, sample with four closely spaced laser foci Analysis, GeneGo Meta Core and CLC them to act as a ‘glue’ that binds cells together. especially for the use of photo-activation, instead of one. Continuing our successful use of Genomics Workbench, which are available to all This challenging project has matured in recent -bleaching, and –switching; total internal adaptive optics on the OPO beam path of our researchers at the Beatson, we use the months and we are currently performing reflection fluorescence (TIRF) microscopy; old TRIM microscope, the new system will have Oncomine Research Premium Edition database parameter studies to determine regions of the intravital microscopy; and fluorescence lifetime a developmental adaptive optics unit built into to satisfy the increasing demands of researchers model’s parameter space that produce imaging for the determination of fluorescence the scanhead. In partnership with M Squared who wish to exploit publicly available datasets. agreement between simulated and resonance energy transfer (FLIM-FRET). More Lasers, we were awarded a Technology Strategy experimentally observed behaviour. Once the recently PET, SPECT and CT imaging were Board project in July entitled ‘Optimised Lasers In 2013 we focused on improving and model has been calibrated, we intend to use it to identified as techniques having great potential to for Multicolour Multiphoton imaging’ that will expanding our service, particularly in the fields test various researcher-defined hypotheses that support the extensive use of preclinical cancer explore the use of semiconductor disk lasers as of next generation sequencing and specifically relate to E-cadherin. models at the Beatson. Collectively we provide alternative excitation sources for two-colour mathematical modelling. To develop our skills in our users with a powerful technology toolbox multiphoton imaging. Finally, much time and working with sequencing data, we attended the We are pleased that there is a constant interest in for cellular and molecular level investigations of effort this year went into ‘shaking down’ the EMBO Practical Training Course on Analysis of our services, and we will continue our efforts to disease and response to therapy in vitro and in Albira and the Andor Spinning Disk systems. The High-Throughput Sequencing Data. Our team deal with all incoming projects effectively and vivo. Andor system has suffered from software also expanded to three in January with the within a reasonable timeframe. Our aim is to problems and some alignment instability but is arrival of Matthew Neilson, who has since ensure that we provide Beatson researchers The BAIR staff serve as a repository of expert now performing well and has been employed his expertise in mathematical with the best possible service and we welcome knowledge who train users and assist with enthusiastically embraced for live imaging of modelling to embark on several modelling and any opportunities to offer advice on topics such advanced applications. We install, maintain, actin dynamics in Dictyostelium. simulation-based projects. as experimental design, statistical troubleshoot and manage the repair of methodologies and data presentation. equipment, serving as an important link Obstacle detection has been incorporated into our existing computational model for Publications listed on page 80 pseudopod-centred cell migration and

48 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 49 METABOLOMICS PROTEOMICS AND MASS SPECTROMETRY www.beatson.gla.ac.uk/advanced_technologies www.beatson.gla.ac.uk/advanced_technologies

The Metabolomics facility forms part of the Beatson’s new The facility has five mass spectrometers used for both Cancer Metabolism Research Unit (headed by Eyal Gottlieb) proteomics and metabolomics: two Orbitrap Velos mass and, in addition, is involved in collaborations with several other spectrometers (one of which is used by Sara Zanivan’s group) research groups at the Institute. Our aim for the future is to be coupled to Easy-LC II nano HPLCs; two Exactive mass able to measure all the low molecular weight metabolites spectrometer systems for metabolomics (operated by Gillian present in the human body. The Human Metabolome Mackay, see page 50); and an AB-Sciex 5600 triple TOF system. Database currently contains over 40,000 metabolites. Head Head The Orbitrap Velos systems are primarily used any of these software packages.This year, in for protein and peptide identification, post- collaboration with Robert Insall’s group, we Gillian Mackay We concentrate on targeted metabolomics, Working together with the Cancer Metabolism Nick Morrice translational modification analysis and have set up lipidomic analyses for LPAs, LPCs, analysing for specific known metabolites, to Research Unit, we have determined metabolic Scientific Officer Research Scientist quantitative proteomics using SILAC. The 5600 PAs, DAGs and, to a more limited extent, other determine the relative quantification between flux in cell culture experiments. Using labelled Niels van den Broek Emma Carrick Triple TOF is used for quantitative proteomics phospholipids on our 5600 Triple TOF system. experimental and control groups as well as carbon or nitrogen tracers, such as 13C glucose, using SILAC, iTRAQ or label-free analyses. We The primary assay has been to develop a very examining metabolic flux. We can also perform we can detect the different isotopes of many Scientific Officers Sergio Lilla have also introduced data independent LC-MS quick analysis of LPAs in media in which untargeted metabolomics, producing a detailed intracellular metabolites over time. We have David Sumpton using SWATH technology and parallel reaction melanoma cells have been grown. These cells metabolic profile that can be used to look for accurately quantified specific metabolite monitoring (pRM) using the Orbitrap Velos are now known to show chemotaxis towards novel metabolic changes. concentrations and calculated intracellular system, where peptide ions of interest are LPA gradients and measuring the individual LPA isotopomer levels (nmol/µg protein) at different constantly selected for MS/MS and then levels has become a high priority. An example of For metabolomics projects, we provide time points. Quantification of the concentration quantified using Skyline software from the this type of analysis is shown in Figure 1. research groups with advice on study design, of metabolites in cell culture medium over time MacCoss group in Seattle. This approach is at sample preparation, data analysis and has enabled us to calculate the rate of uptake least two orders of magnitude more sensitive The facility focuses on four major areas: quantification of metabolites. The scope of or secretion of metabolites by cells from the than LC-MS using conventional data dependent our current analyses includes amino acids, medium. This extracellular metabolite 1. Quantification of protein complexes, acquisition (DDA) and has been used organic acids, sugars, sugar phosphates and exchange rate is a key parameter for metabolic proteomes and secretomes using label and successfully by our group to quantify proteins label-free methods. nucleotides, although other small polar flux modelling. and phosphopeptides that were ‘invisible’ in the molecules can be detected in the same screens. 2. Post-translational modification analysis of DDA analysis. In August, we purchased an Agilent GC-MS/MS individual proteins, in particular We have two members of staff in the core system, and have developed a promising phosphorylation, acetylation, methylation The experienced staff members provide and ubiquitination. Recent research has facility, and two other research scientists, from method for fatty acid analysis. We are also advanced knowledge and expertise for initiation involved the chemical modification of Eyal Gottlieb and Karen Vousden’s labs, who currently developing other GC-MS methods for of proteomics projects and state-of-the-art proteins by naturally occurring metabolites use the mass spectrometers for their own work a range of metabolites. With our Exactive proteomics analyses to the Institute’s scientific such as succinylation of cysteine/lysine and occasionally for other specific projects in LC-MS, we are investigating UPLC analysis to community. It is advisable to contact one of the and kyneurinin modification of cysteine/ their groups. dramatically reduce analysis time for some of staff members prior to initiating a project so that histidne/lysine. our standard methods, as well as developing the best experimental design can be drawn up. 3. Lipidomic profiling of known, naturally We have two Thermo Scientific Exactive LC-MS LC-MS methods for fatty acid analysis. A Although most of the activity of the service is occurring phospholipids such as LPA, LPC, systems for metabolite analysis. The Exactive Q-Exactive system has been purchased and will related to the analyses of samples for protein PA, PS, PI, PC and cardiolipin. mass spectrometer has a polarity-switching be installed in January 2014, to enable LC-MS/ identification and characterisation by MS, the feature, allowing many metabolites to be MS analysis for the identification of unknown 4. Method development for proteomic and analysis of post-translational modifications, detected in the same run. Our focus on metabolites, and provide structural information small molecule analysis. especially protein phosphorylation, is a glycolysis and the tricarboxylic acid cycle has on compounds. specialty. Along with the investment in MS resulted in robust LC-MS methods for Publications listed on page 82 instrumentation, we are expanding the metabolites of these pathways. We have Publications listed on page 82 informatics capability of the facility. There is expertise in Thermo’s data analysis package Figure 1 now a dedicated MaxQuant server for the LCquan for targeted metabolomics, and for LC-MS analysis of analysis of SILAC data, a new Mascot server, untargeted data analysis we use Thermo’s Sieve lysophosphatidic acids. Proteome Discoverer 1.4, Progenesis 4 and software. Skyline for the analysis of label free/SWATH or pRM data. Data can be disseminated to researchers using Scaffold Q + S, which has a free, user friendly viewer. Training is always available from the facility staff on how to use 50 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 51 RNAi SCREENING TRANSGENIC MODELS OF CANCER www.beatson.gla.ac.uk/advanced_technologies www.beatson.gla.ac.uk/advanced_technologies

The RNAi Screening facility couples high throughput RNA The Transgenic Models Laboratory uses in vivo models to interference (RNAi) screening with high content imaging (HCI) accurately recapitulate human cancer in a physiologically to translate fundamental cancer research towards new relevant way to better understand how cancers develop and therapies. We can sequentially knockdown every gene in the metastasise. With sophisticated preclinical models we can genome and quantify its effect on a biological system. This assess the genetic causes of cancer allowing us to develop approach is a powerful tool for elucidating new interactions biologically relevant systems for testing novel therapies. The and identifying novel drug targets and/or drug interacting lab has a particular interest in the RUNX gene family and is Head partners to improve existing cancer therapeutic approaches. Head investigating the role of RUNX1 and RUNX2 in breast and Emma Shanks Karen Blyth prostate cancer. Scientific Officer Over the past two years, the facility has Over the last year, we have supported the Research Scientists Lynn McGarry Kirsteen Campbell1 collaborated with nine groups and completed acquisition of a number of bespoke siRNA Laura McDonald Preclinical genetic models have significantly the most frequently altered genes in leukaemia, 17 screens, including 11 in 2013. Notable collections designed to target specific Informatics Manager contributed to our understanding of how where loss of function caused by chromosomal Daniel James projects in the past year include identification of processes, in particular a metabolism focused Scientific Officers cancers develop and metastasise, and the role translocation and mutation predisposes to adult genes that are synthetic lethal with i) KrasG12D/ one of 1,800 genes. We also offer access to Dimitris Athineos of the Transgenic Models lab is to exploit these myeloid and childhood lymphoid leukaemia. Graduate Student p53-/- in a pancreatic cancer cell line (Owen three libraries of clinically tested/approved drugs Susan Mason Matthew Davidson cancer models to address important biological Recent evidence suggests these genes may also Sansom), ii) MYC expression in U2OS cells for a wide range of indications unrelated to Clinical Research Fellow questions in a physiologically relevant in vivo have a role in breast cancer. In collaboration (Daniel Murphy) and iii) miR21 in colorectal cancer, which are used to support a drug 2 Anne McKillop setting. In collaboration with other Beatson with researchers at the University of Glasgow, cancer (Nicola Valeri). We also conducted an re-purposing approach. As the principal targets research groups, we are using genetic and we have been assessing archival human breast investigation into drugs that are synthetically of these drugs are established, we often overlay Graduate Students transplantation models to assess the signalling cancer tissues for expression of RUNX1 and lethal with radiation exposure in glioblastoma. these data with siRNA screening datasets to Nicola Ferrari Alessandra Riggio pathways and genetic mutations that are RUNX2. We find that overexpression of RUNX1 in isolate the most pertinent targets for validation. associated with cancers such as pancreatic, a specific subtype of human breast cancer, the Viability based on nuclear count is our most To date, we have conducted drug re-purposing Intern colorectal, breast and prostate cancer, and so-called triple negative (ER/PR/HER2-negative) requested output, yet HCI facilitates screens in pancreatic cancer, glioblastoma and Sinead Griffin3 melanoma. These cancer models, which breast cancers, correlates with poorer survival quantification of a suite of phenotypic breast cancer models, the latter being funded 1 faithfully recapitulate the genetic regulation, (Fig. 1) and may be an independent prognostic parameters pertaining to nuclei and/or cells. For by Breast Cancer Campaign. Royal Society 2 sporadic development, tumour pathology and marker for this patient group. This is particularly example, nuclear size can be indicative of CRUK Glasgow Centre (joint with School of organ-specific metastatic spread of specific important, as there is currently no good cellular senescence or the scattering of cells Finally, we welcomed graduate student Veterinary Medicine) human epithelial cancers, are particularly biomarker or targeted therapy for this subtype of may be suggestive of a metastatic phenotype. Matthew Davidson whose project will utilise 3 University of Glasgow valuable for monitoring responses to breast cancer. We also find RUNX2 to be We now routinely offer quantification of siRNA screening and drug re-purposing to therapeutic agents in our efforts to develop overexpressed in some triple negative breast additional phenotypic parameters to maximise identify and validate innovative therapeutics for Figure 1 improved and novel targeted therapies to the cancers and indeed have evidence that Runx2 the return on the investment of conducting a the treatment of oral/oropharyngeal cancers. RUNX1 may be a new prognostic common cancers. may be specifically involved in the squamous screen. Moreover, with the implementation of The facility also relocated into the Wolfson Wohl marker in triple negative breast metaplastic form of the disease. Interestingly the Columbus Image Data Storage and Analysis Cancer Research Centre to provide some much cancer. Immunohistochemistry RUNX1 and RUNX2 in breast and RUNX2 is also expressed in the stromal System (Perkin Elmer), we have the additional needed additional space. images demonstrating high prostate cancer component of breast cancers where it flexibility to conduct machine learning, where levels of RUNX1 in human breast cancer tissues. High RUNX1 RUNX1 and RUNX2, which are essential for correlates with worse survival. Current projects we can manually teach software what Future developments expression is associated with normal mammalian development, are genes in the lab are looking at the role of Runx2 as a phenotype(s) are of interest and then apply the To meet increasing demand, we have poorer overall survival in patients that are associated with cancer. RUNX1 is one of regulator of mammary stem and/or progenitor resulting algorithms to an entire screening purchased an additional Operetta HCI system, with triple negative breast cancer cells, the role of Runx1 and Runx2 in the dataset. This is a very powerful way to conduct which has confocality. This will increase our as shown by the survival graph. maintenance of mammary epithelium phenotypic analysis. We have also been working ability to conduct more challenging screens and homeostasis and the dysregulation of these with Owen Sansom’s group to develop our improve our capacity to support validation genes in epithelial cancers, in particular in breast three-dimensional quantification capabilities. experiments through more detailed imaging and prostate cancer. Currently, we are able to quantify cancer cell acquisition and analysis. Further to this, we aim spheroids using our metabolic, fluorometric to develop our aptitude in conducting screens Publications listed on page 76 screening assay and are developing an within a three-dimensional environment. image-based assay to complement this.

52 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 53 TRANSGENIC TECHNOLOGY www.beatson.gla.ac.uk/advanced_technologies

The Transgenic Technology Laboratory uses genetic manipulation technology to help understand gene function in cancer. This also allows the generation of new and more BEATSON accurate models of human cancers. By the use of gene targeting in stem cells and direct genome editing we can precisely introduce defined alterations of specific genes into ASSOCIATES the germline, allowing us to create increasingly sophisticated Head cancer models. Douglas

Strathdee Using stem cells to model cancer culture to produce a wide variety of cell types. Scientific Officers Embryonic stem cells have a number of So, the consequence of the mutation can be Laurence Cadalbert properties that make them an extremely useful analysed not only in stem cells themselves but Farah Naz Ghaffar tool for the analysis of gene function. One of the also in a variety of specialised cell types derived most important of these is their high rate of DNA from them. Graduate Student Nicola Laprano1 recombination, termed homologous recombination. We can exploit this property to Employing state-of-the-art genetic 1 from October engineer very defined alterations of target genes manipulation techniques to allow us to interrogate the function of these In addition to maximising the potential of genes in cells. These can be, for example, existing technologies, we are interested in knockout alleles where the function of a specific implementing new technologies with the aim of gene is compromised. This allows direct refining and improving current cancer models. assessment of the role of the target gene in In addition to standard knockout and transgenic specific cellular processes. Also, we can use the approaches, we are testing new approaches to same technology to introduce point mutations allow us to extend the range of projects we can Figure 1 that mimic the exact changes uncovered in the consider. To this end we are currently testing UNIVERSITY OF GLASGOW A screen for gene involved in analysis of human cancers. Taken together shRNA methods, designed to allow us to control regenerating intestinal epithelium. The number of cells these techniques allow us to carefully the level of gene expression in tumours directly. that are responsible for interrogate the role of genes in cell and tissue Methods that allow us to regulate the level of a Peter D. Adams - Epigenetics of Cancer and Ageing populating the intestinal homeostasis and the development of cancer. protein in this manner should enable, for epithelium are assessed in We collaborate on a wide variety of different example, the validation of candidate genes as Daniel J. Murphy - Oncogene-Induced Vulnerabilities controls (A). Phenotypes can be projects and use a number of strategies, such potential targets for drug development. In observed in samples lacking the Stephen Tait - Mitochondria and Cell Death function of particular genes as point mutations or conditional knockouts, collaboration with the Sanger MGP (Mouse where the number of cells in the to produce finely controlled alterations in gene Genetics Project) and the IMPC (International crypts is reduced (B) or where activity. Once the desired genetic alteration has Mouse Phenotyping Consortium), we are the number of cells is greater been introduced into stem cells, the cells can currently establishing a screen to analyse the than the controls (C). Genes be phenotyped to assess the consequence of role of a variety of genes in intestinal identified as playing a role in intestinal regeneration represent the desired mutation on the biology and homeostasis (Fig. 1). Candidate genes are good candidate genes for a behaviour of the cells. One advantage of using identified and the function of these in potential role in tumourigenesis. stem cells is that they can be differentiated in maintaining cells populations in the intestine is assessed. The aim of this work is to identify candidate genes that may play a role in the maintenance of cell number in the intestinal epithelium and are therefore good candidate genes that may play an important role in tumour formation in the intestine.

Publications listed on page 86

54 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE BEATSON ASSOCIATES 55 Figure 2 chromatin insulator and an ATP-dependent EPIGENETICS OF CANCER AND AGEING DNA methylation gains and losses chromatin-remodelling complex. Our results in senescent cells overlap with map the distribution of the HIRA chaperone www.gla.ac.uk/researchinstitutes/cancersciences/ hyper- and hypomethylation in cancer. Paradoxically, this across the chromatin landscape and point to staff/peteradams/ suggests that the epigenome of different interacting partners at functionally senescent cells might be primed distinct regulatory sites (Pchelintsev et al., Cell for progression to cancer. In turn, Rep 2013; 3: 1012). this suggests that senescence might be an imperfect tumour suppressor mechanism, and These studies are being extended to an accumulation of senescent cells analysis of HIRA, histone H3.3 and other with age might predispose to chromatin features in senescent cells. We have cancer. The reason(s) why cancer Figure 2 performed ChIP-seq and DNA methyl-seq of increases with age in humans is several histone modifications and DNA The Adams lab investigates the impact of chromatin structure poorly understood at present (Cruickshanks et al., Nat methylation in proliferating and senescent cells and epigenetics on cell proliferation, ageing and cancer. In Cell Biol 2013; 15: 1495). (e.g. Shah et al., Genes Dev 2013; 27: 1787). particular, we hypothesise that age-associated changes in These studies have revealed remarkable and Figure 3 paradoxical insights into chromatin in The altered epigenome of chromatin structure, function and regulation contribute to the senescence, and the role of senescence as a senescent cells might promote an dramatic age-associated increase in the incidence of cancer. age-associated increase in the tumour suppressor and its contribution to tissue incidence of human cancers. Our ageing. Mechanistic hypotheses are being While age is the biggest single risk factor for most cancers, the analyses and previous studies and tested, and these analyses are being extended to ideas lead to the following model: studies of human and mouse aged and pre- reason for this is current poorly understood. Senescent cells accumulate with Group Leader malignant tissues to define the mechanism by age in human tissues. These Peter D. Adams senescent cells harbour low-level which age-associated chromatin changes Cell senescence is an irreversible proliferation chromatin structure, and onset of diseases of methylation of CpG islands of predispose to cancer. Beatson Associate arrest instigated by a variety of molecular ageing, including cancer. tumour suppressor genes that is triggers including acquisition of activated insufficient to silence gene Research Scientists Senescent cells appear ‘epigenetically expression. However, even a Dina Dikovskaya oncogenes, and shortened telomeres caused Genome-wide analysis of chromatin Figure 3 primed’ to form cancer cells limited/transient escape from Andrejs Ivanov by excess rounds of cell division. In addition, structure and chromatin regulators in senescence, for example due to Altered DNA methylation and associated Tony McBryan senescent cells secrete a cocktail of senescent cells genetic inactivation of PTEN, sequencing. To complement this analysis of destabilisation of genome integrity and function David Nelson inflammatory cytokines, chemokines and matrix To better understand the structure and function confers additional rounds of cell is a hallmark of cancer. Replicative senescence Jeff Pawlikowski epigenetic marks in senescence, we are also proteases (the ‘inflammatory secretome’ or of chromatin in senescent cells, we are division that permit proliferation- Nikolay Pchelintsev exploring the genome-wide distribution of is a tumour suppressor process that imposes a dependent spreading of DNA Taranjit Singh Rai senescence-associated secretory phenotype, performing genome-wide analyses of histone limit on the proliferative potential of normal cells methylation. Hence, an initial histone chaperones in senescent cells, again John van Tuyn SASP) that is capable of influencing behaviour of modifications and DNA methylation to compare ‘seed’ of methylation in a using state-of-the-art approaches. In addition, that all cancer cells must bypass. Here we show neighbouring cells, including immune cells. chromatin in proliferating and senescent cells. senescent cell in an aged tissue we have collected gene expression data to build by whole-genome single-nucleotide bisulfite Clinical Research Fellows Compelling evidence now indicates that cell To do this, we are using next generation can, in conjunction with other sequencing that replicative senescent human Annie Latif a comprehensive, integrated view of the senescence is a potent tumour suppression sequencing (ChIP-seq), microarray and genetic alterations and clonal Douglas MacKenzie epigenetic control of senescent cell function. cells exhibit widespread DNA hypomethylation selection, grow to full CpG island mechanism, notably in cells harbouring proteomic approaches and whole genome and focal hypermethylation. Hypomethylation hypermethylation and tumour Scientific Officer activated oncogenes. Senescence-associated single-nucleotide bisulphite modified DNA suppressor gene silencing, To initiate studies in this area, we have analysed occurs preferentially at gene-poor, late- Claire Brock proliferation arrest and the SASP act in concert thereby facilitating progression to the HIRA histone chaperone complex. This replicating, lamin-associated domains and is to achieve tumour suppression: proliferation late-life cancer. Global linked to mislocalisation of the maintenance Graduate Students complex, comprised of HIRA, UBN1 and arrest directly curtails tumour growth and the hypomethylation in senescent Rachael Hewitt CABIN1, collaborates with histone-binding DNA methyltransferase (DNMT1) in cells cells may also promote genome Indrani Manoharan SASP calls on innate immune cells to eliminate approaching senescence. Low-level gains of instability and dysfunction protein ASF1a to incorporate histone variant Desiree Piscitello1 the offending damaged cells. Because of (Cruickshanks et al., Nat Cell Biol H3.3 into chromatin in a DNA replication- methylation are enriched in CpG islands, senescence, most primary human cells have a 2013; 15: 1495). independent manner. Consistent with this role including at genes whose methylation and 1 joint with David Gillespie finite proliferative lifespan, and evidence has in DNA replication-independent chromatin silencing is thought to promote cancer. Gains been presented that senescence contributes to metabolism, we have previously implicated this and losses of methylation in replicative tissue ageing in vivo, in part by limiting the chaperone in the regulation of chromatin in senescence are thus qualitatively similar to proper self-renewal of stem cells and tissues. non-proliferating cells. To better understand those in cancer (Fig. 2), and this ‘reprogrammed’ In sum, cell senescence has both beneficial HIRA’s function and mechanism, we integrated methylation landscape is largely retained when and detrimental effects, and bypass of HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq cells bypass senescence. Consequently, the senescence can lead to tumour formation and gene expression analyses. Most HIRA- DNA methylome of senescent cells might through uncontrolled proliferation of Figure 1 binding sites co-localise with UBN1, ASF1a and promote malignancy, if these cells escape the damaged cells (Fig. 1). Senescence as a tumour suppressor mechanism. H3.3 at active promoters and active and weak/ proliferative barrier. Since senescent cells appear Acquisition of an activated oncogene or inactivation of a poised enhancers. At promoters, binding of to be imperfect tumour suppressors, at least Cellular senescence, ageing and cancer are all tumour suppressor initially causes a proliferative burst. HIRA/UBN1/ASF1a correlates with the level of from an epigenetic perspective, accumulation accompanied by marked changes in chromatin Ultimately, senescence kicks in to arrest proliferation of the cells harbouring the oncogenic event. Proliferation gene expression. HIRA is required for deposition of senescent cells in aged tissues might structure. We are interested in the epigenetic arrest is reinforced through the senescence-associated of histone H3.3 at its binding sites. There are contribute to increased incidence of cancer changes associated with senescence, and their secretory phenotype (SASP). Senescence-associated marked differences in nucleosome and co- with age (Fig. 3) (Cruickshanks et al., Nat Cell Biol contribution to the senescent phenotype. In proliferation is likely to arrest tumour progression by regulator composition at different classes of 2013; 15: 1495). addition, since senescent cells promote ageing, preventing proliferation of neoplastic cells and HIRA-bound regulatory site. Underscoring this, we are testing the hypothesis that senescence- suppressing accumulation of additional genetic alterations. In addition, senescence recruits the innate we report novel physical interactions between Publications listed on page 88 associated changes in chromatin structure immune system to clear the genetically altered cells that the HIRA complex and transcription factors, a contribute to age-associated changes in threaten the host with malignant disease.

56 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE EPIGENETICS OF CANCER AND AGEING 57 ONCOGENE-INDUCED VULNERABILITIES www.gla.ac.uk/researchinstitutes/cancersciences/ staff/danielmurphy/

Oncogenic signalling profoundly alters how cells respond to their environment, typically putting tumour cells under tremendous pressure to reconcile conflicting cues. For example, tumour cells must re-organise their metabolic pathways to balance competing needs for biosynthetic precursors with energetic homeostasis, often while surviving in a milieu of limited oxygen and nutrients. Our overarching Group Leader hypothesis is that such oncogene-induced biological Daniel perturbations can be exploited for cancer therapy, even in J.Murphy Beatson Associate the absence of direct suppression of driver oncogenes. We use deregulated MYC as our paradigm oncogene coupled Research Scientist Meera Raja with a mixture of candidate and RNAi-based screening

Scientific Officer approaches to identify induced vulnerabilities in vivo and in Alan McVie vitro, and are actively exploring several strategies for selective Graduate Students Tiziana Monteverde elimination of cells that overexpress MYC. Nathiya Muthalagu Figure 1 Sarah Neidler molecular basis of the role of ARK5 in bio- This year was focused on integrating into our Induced dependencies need Jennifer Port MYC in cancer failed to return predicted cooperating energetic homeostasis as well as to genetically new environment and establishing the tools not reflect direct molecular model the therapeutic impact of ARK5 and collaborations necessary to drive our Overexpression of the transcription factor MYC oncogenes such as RAS or PI3K. Instead, we interactions. Oncogene-induced occurs in a huge number of human cancers identified a number of metabolic regulators, cell growth, typically requiring suppression on MYC-dependent tumours research programme forward. The lab arising from almost every tissue type. MYC including the AMP-activated protein kinase, signal transduction via the in vivo. attracted competitive funding from the British overexpression may arise from focal or broad AMPK and the closely related ARK5 (aka NUAK1), MTOR (mechanistic target of Lung Foundation, European Union and CRUK chromosomal amplification, gene as required for viability specifically when MYC is rapamycin) pathway, drives MYC-induced lung cancer progression Centre Development Fund, as well as entering a rampant ATP consumption that Lung cancer remains one of the deadliest funded collaboration with Merck Sharp & translocation, enhanced mRNA and protein overexpressed. Why these kinases? MYC-driven must be compensated for stability or indeed increased signalling through biosynthetic processes cause cells to consume through increased cellular intake forms of cancer worldwide, accounting for Dohme, together enabling us to welcome four upstream regulatory factors such as RAS, ATP at a much higher rate than normal and, of fuel (e.g. glucose, amino and some 18% of all cancer related deaths, and new lab members, including two graduate NOTCH or beta-catenin. In a number of in vivo despite elaborate reorganisation of carbon fatty acids) combined with incidence of lung cancer is on the rise especially students. We have generated a number of settings, MYC overexpression is sufficient to metabolism to balance ATP production with AMPK-mediated attenuation of in the increasingly industrialised and densely exciting preliminary results to build upon and macromolecular synthesis. populated cities of emerging economies. look forward to a highly productive and initiate or exacerbate tumourigenesis and biosynthetic precursor provision, this engenders Upon suppression of ARK5, this moreover is typically required to sustain the a state of energetic stress that leads to AMPK feedback mechanism is Poor prognosis arises in large part from the successful 2014. cancerous phenotype. A successful therapeutic activation. AMPK in turn inhibits TORC1 to impaired, leading to ATP combination of late disease detection and strategy that exploits MYC overexpression attenuate the rate of macromolecular synthesis, depletion and bio-energetic limited matching of patients with emerging would likely have a tremendous impact on effectively allowing cells to balance the rate of catastrophe. In principle, any targeted therapies. We have developed a intervention that similarly impairs model for early tumour progression using human health. ATP consumption with production. For reasons bio-energetic homeostasis may that are presently unclear, ARK5 is required for selectively kill tumour cells. tractable combinations of conditional alleles, MYC-induced metabolic vulnerability this homeostatic feedback mechanism. including Kras and MYC. We are using laser- Rather than attempting to target MYC directly, a Depletion of either AMPK or ARK5 thus leads to capture microdissection combined with gene number of groups including our own have ATP collapse and consequently loss of viability, expression analysis and functional screening turned to ‘synthetic lethal’ screening selectively in cells overexpressing MYC, strongly to identify proteins with enzymatic activities approaches in order to identify induced suggesting that targeting these kinases may be that are required for progression from benign vulnerabilities, i.e. proteins and pathways to therapeutically effective against MYC to malignant disease. The dual goal of this which cells overexpressing MYC – but, crucially, overexpressing cancers. In 2013, the lab endeavour is to identify both new candidate not normal cells – are addicted for survival. Our developed and acquired a number of key tools therapeutic targets and surrogates for siRNA screen of the human kinome surprisingly that will now enable us to understand the early detection.

58 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE ONCOGENE-INDUCED VULNERABILITIES 59 allows mitochondrial repopulation. We found MITOCHONDRIA AND CELL DEATH that the ability of certain mitochondria to evade www.gla.ac.uk/researchinstitutes/cancersciences MOMP is due to increased anti-apoptotic Bcl-2 protein expression on their outer membrane. /staff/stephentait/ Following from this, treatment with BH3-mimetic compounds effectively induced complete MOMP and prevented clonogenic survival, a result that may have therapeutic relevance. Current work is aimed at addressing the importance of post-MOMP cell survival in tumourigenesis and treatment response using a variety of approaches including developing a Cell death is a potent tumour suppressor mechanism that must means to report cell survival following MOMP in vivo. Secondly, we are investigating the be evaded in order for cancer to develop. Because anti-cancer mechanisms that enable cell survival, focusing therapies often act by killing cells, cell death sensitivity also upon the protective role of autophagy in this governs therapeutic efficacy. Paradoxically, besides powering process. our cells, mitochondria are essential for apoptosis, the major Mitochondria and non-apoptotic cell death Various other forms of programmed cell death form of programmed cell death. We aim to understand how exist besides apoptosis, including caspase mitochondria regulate cell death and define how this process is independent cell death, necroptosis and mitotic Group Leader deregulated in cancer. Our goal is to clinically translate these cell death. These alternate forms of cell death Stephen Tait function as either back-up pathways in the event of failed apoptosis or are distinct cell Royal Society University findings in order to improve existing anti-cancer therapies and death programmes in themselves. Although Research Fellow develop new means to selectively kill cancer cells. Beatson Associate these cell death modalities are likely important in cancer (particularly in response to therapy) Research Scientists little is known about their underlying molecular Mitochondria, cell death and cancer binding Bax and Bak (the effector proteins that Martina Haller Figure 1 mechanisms. Mitochondria have been Gabriel Ichim1 Apoptosis requires caspase protease cause MOMP). Our recent data supports a implicated as major effectors in necroptosis, a Maria Karvela activation leading to widespread substrate unified model; under conditions of low form of regulated cell death that requires cleavage and rapid cell death. During apoptotic stress, Bcl-2 proteins inhibit MOMP by Graduate Student activation of the kinase RIPK3. We have directly apoptosis, mitochondrial outer membrane binding BH3-only proteins and under high levels Evangelos Giampazolias2 tested a role for mitochondria in necroptosis by permeabilisation (MOMP) occurs, a crucial of stress they block MOMP by binding activated generating mitochondria-free cells through Visitor event that is required for caspase activation. Bax and Bak. Cells in which anti-apoptotic Bcl-2 Parkin-mediated mitophagy (Fig. 1). Using this Jonathan Lopez Following MOMP, mitochondrial inter- proteins suppress MOMP by binding active Bax method, we find that the kinetics and extent of membrane space proteins, such as or Bak are much more resistant to the addition 1 EMBO necroptosis are not affected by mitochondrial cytochrome c, are released into the cytoplasm of BH3-mimetic compounds, a result that may 2 CRUK Glasgow Centre depletion thereby ruling out a major role for where they drive caspase activation and have important clinical implications. We are mitochondria executing necroptosis (Fig. 2). Our apoptosis. Given its key role in controlling cell currently investigating the relevance of these currents studies focus upon investigating if survival, mitochondrial outer membrane findings in a therapeutic context. mitochondrial dysfunction can serve to initiate integrity is highly regulated, largely through necroptosis in addition to defining the role for interactions between pro- and anti-apoptotic Cell survival following mitochondrial necroptosis in tumour suppression and therapy- Bcl-2 proteins. Cancer cells commonly inhibit permeabilisation induced cell death. apoptosis by preventing MOMP, often through Following MOMP, cells often die irrespective of upregulation of anti-apoptotic Bcl-2 proteins or caspase activation, suggesting that MOMP Publications listed on page 88 by inhibiting caspase activity downstream of represents a ‘point-of-no-return’. However, we MOMP. Newly developed anti-cancer therapies have found that cells can sometimes survive target these apoptotic blocks. For example, and proliferate following MOMP. This may be BH3-mimetic compounds exploit the Bcl-2 important in cancer development and addiction of certain cancer cells leading to regulating therapeutic sensitivity as cancer cells Figure 2 tumour specific killing. often display defective caspase activity. We determined that survival following Figure 1 Regulating the mitochondrial gateway to mitochondrial permeabilisation was dependent, Defining mitochondrial functions through Parkin-mediated mitophagy. Top: Three- dimensional EM showing mitochondrial depletion in Parkin-expressing cells specifically death at least in part, upon glycolysis and autophagy. following CCCP treatment (mitochondria are in red, cytosol is in blue). Bottom: Using this The mechanism of MOMP inhibition by Bcl-2 More recently, using novel live cell imaging approach, we can address the importance of mitochondria in cell death and other processes proteins is controversial. Two prominent models techniques, we have discovered that MOMP can by comparing mitochondria proficient and deficient cells. have been proposed: Bcl-2 proteins inhibit be incomplete such that some mitochondria MOMP either through binding BH3-only remain intact. Intact mitochondria are required Figure 2 RIPK3-dependent necroptosis does not require mitochondria. 3T3-SA cells expressing vector proteins (a subfamily of Bcl-2 proteins that relay for cell survival following MOMP probably by or Parkin were treated with or without CCCP then treated with TNF/zVAD to induce the apoptotic signal to the mitochondria) or by serving as a source of healthy mitochondria that necroptosis. Cell death was measured by Sytox Green uptake in an Incucyte imager. No difference in the kinetics or extent of necroptosis was observed between mitochondria in replete versus depleted (Parkin + CCCP) cells.

60 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE MITOCHONDRIA AND CELL DEATH 61 RESEARCH GROUPS

UNIVERSITY OF GLASGOW

Jeff Evans - Institute of Cancer Sciences

UNIVERSITY OF GLASGOW 63 INSTITUTE OF CANCER SCIENCES www.gla.ac.uk/researchinstitutes/cancersciences/

The Institute of Cancer Sciences (ICS) at the University of intestine to understand how cell autonomous therapeutic target because treatment options and niche-derived signals integrate to regulate are so limited in this disease. Glasgow spans fundamental cancer biology, translational stem cell proliferation in response to damage as and clinical cancer research with a major focus on cancer well as tumourigenesis of adult self-renewing A collaborative study with Cold Spring Harbor tissues. Laboratories and the Memorial Sloan-Kettering genomics and disease-specific research. The ICS also spans Cancer Centre has shown that inhibition of three campuses, at the Garscube Estate (adjacent to the CRUK Cell death is a key tumour suppressor signalling through PDGFRβ can block mechanism that must be inhibited in order for metastasis promoted by mutations in the TP53 Beatson Institute), Gartnavel (including the Paul O’Gorman cancer to develop, and sensitivity to cell death gene, which are common in pancreatic cancer. Leukaemia Research Centre [POG-LRC] and the Beatson West also governs therapeutic efficacy. Stephen High PDGFRβ expression correlates with poor Tait’s group (see page 60) is focused on disease-free survival in pancreatic, colon and Director of Scotland Cancer Centre) and the New South Glasgow Jeff Evans understanding how mitochondria control ovarian cancer patients, implicating PDGFRβ as Hospital. Its primary goal is to deliver world-class research apoptotic cell death and addressing how this is a prognostic marker and possible target for deregulated in cancer. Similarly, cell immortality attenuating metastasis in p53 mutant tumours. that can be translated to patient benefit and to provide a is one of the hallmarks of cancer development leading-edge environment for research and training. and is directly controlled by telomerase. The groups also contributed to a major Research in Nicol Keith’s laboratory has international catalogue of somatic mutations in demonstrated the validity of this target through cancer genomes. The results reveal the diversity This year, the ICS was a major partner in the cells. The malignant properties of cancer cells diverse telomerase targeting approaches. The of mutational processes underlying the successful renewal of the CRUK Glasgow have their origins in DNA mutations and other lab has discovered key targets that regulate development of cancer, with potential Centre, and a major contributor to the forms of genetic damage but also in epigenetic telomere homeostasis and is translating these implications for understanding of cancer University’s Research Excellence Framework changes. These are heritable through cell findings on telomerase, cell senescence and aetiology and prevention. Importantly, they also (REF) submission including a number of impact division but are not directly coded in the DNA. telomere dysfunction into biomarker clinical identify mutational patterns that may provide a case studies. Notable achievements in 2013 Instead, they are coded within chromatin, studies in patients. Paul Shiels’ group has also basis for a molecular phenotype-guided included the completion and occupation of the through DNA methylation, non-canonical developed and validated biomarkers of ageing therapeutic strategy, in which next generation Wolfson Wohl Cancer Research Centre histone variants, histone modifications, histone for use in basic research and translational sequencing is used to screen for mutations that (WWCRC) building. We also continued our binding proteins and higher order patterns of applications. Specifically, they demonstrated characterise cancers that are vulnerable to ambitious recruitment of international research chromatin folding. A number of research groups that the CDKN2 locus could be used to gauge specific existing targeted therapies. leaders. Andrew Biankin took up his post as the (Peter Adams [see page 56], David Vetrie, the impact of non cell-autonomous Regius Chair of Surgery and Director of the Adam West, Katherine West) are interested in senescence in disease states. For example, they Other emerging research interests include WWCRC and Sean Grimmond joined us as basic mechanisms of chromatin regulation, demonstrated that cellular biological age, oncogene-induced vulnerabilities (Daniel Chair of Medical Genomics. We also welcomed epigenetic inheritance and how epigenetic proliferation and immune cell infiltrates were Murphy, see page 58), while Katie Wakeham David Chang (Senior Clinical Lecturer in dysfunction contributes to ageing, cancer and independent prognostic factors in a cohort of is developing a research programme Surgery), Liz Musgrove (Senior Research Fellow response to cancer therapies. For example, a patients with colorectal cancer. investigating the prognostic significance of in Cancer Genomics), Prabs Rajan (CRUK study in the Vetrie and West groups, published in human papillomavirus in vulvar intraepithelial Clinician Scientist, Urological Surgery), Katie Blood, described the chromosomal interactions Pancreatic cancer biology and genomics is a neoplasia, vulvar cancer and anal cancer, and Wakeham (Clinical Lecturer in Clinical that regulate the expression of the TAL1 gene. major research focus for a number of research the prevalence of HPV type-specific genotypes Oncology), while Julia Cordero and Vignir TAL1 is frequently mis-regulated in T-cell acute groups, and with potential clinical application in oropharyngeal cancers diagnosed in the Helgason were both appointed as University lymphoblastic leukaemia (T-ALL). This study (Andrew Biankin, Sean Grimmond, David West of Scotland. Leadership Fellows. We congratulated Mhairi found that chromosomal looping facilitates Chang, Liz Musgrove). Pancreatic cancer is one Copland and Paul Shiels on promotion to both normal and aberrant TAL1 expression and of the most lethal and molecularly diverse Ovarian cancer research is led by Iain McNeish. Professorships, while Kamil Kranc was may predispose to genomic rearrangements malignancies. Re-purposing of therapeutics One major research focus is the biology of appointed to a prestigious position at the that drive T-ALL. that target specific molecular mechanisms in acquired platinum resistance in high grade University of Edinburgh and both Nicola Valeri different disease types offers potential for rapid serous ovarian cancer. The BriTROC project, and Chiara Braconi were appointed to the Stem cells maintain tissue homeostasis by improvements in outcome. Anti-HER2 therapies funded by Ovarian Cancer Action and Cancer Institute of Cancer Research, London. adjusting their function to micro-environmental have a proven track record in the treatment of Research UK, and run in collaboration with or ‘niche’ signals. Deregulation of such signals breast cancer, and the groups have now James Brenton at the CRUK Cambridge Fundamental cancer biology, translational results in uncontrolled stem cell proliferation, established diagnostic criteria for HER2 Institute, is the largest-ever translational study of and clinical cancer research which is often associated with cancer. Julia amplification in pancreatic cancer. Although the relapsed high-grade serous disease. Tumour Major interests of our cancer biologists include Cordero’s laboratory combines studies on the incidence of HER2 amplification in pancreatic biopsies are being acquired at relapse from 300 cellular senescence, ageing, and cancer stem Drosophila midgut and the mammalian cancer is low (2%), it represents an attractive women in 9 UK centres, to allow comparative

64 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE INSTITUTE OF CANCER SCIENCES 65 INSTITUTE OF CANCER SCIENCES (CONTINUED)

genomic analysis of relapsed disease with that carry a dysfunctional BRCA1 gene. One of Recently opened Wolfson Wohl disease in the same patient at the time of the major causes of breast cancer related Cancer Research Centre building diagnosis. The other main research area focuses mortality is the development of endocrine on novel biological therapies, especially resistance. Joanne Edwards has identified IKKα oncolytic virus therapy. Work in 2013 as a target in endocrine resistant breast cancer demonstrated that oncolytic vaccinia induces and is now working in collaboration with death through pathways of programmed University of Strathclyde to assess IKKα inhibitors necrosis and an ongoing collaboration with and identify markers to predict response to Stephen Tait will extend this work to adenovirus these in patients with breast cancer. The group and HSV. In addition, the group demonstrated has extended its studies of endocrine resistance that taxane resistance increases activity of to prostate cancer, having demonstrated that oncolytic adenoviruses through two separate androgen receptor (AR) phosphorylation at mechanisms - increased expression of the serine 81 in response to androgens may predict primary adenovirus receptor CAR and response to Abiraterone, and AR dysregulated cell cycle control. These results phosphorylation at serine 213 by Akt may have shaped the design of the phase 1 OCTAVE predict response to PI3K/Akt inhibitors. The trial (intraperitoneal administration of oncolytic Prostate Cancer Biology group is led by Hing adenovirus ColoAd1 in relapsed ovarian cancer), Leung (see page 16), with Prabs Rajan and which will open in May 2014. Collaboration with Imran Ahmad as junior investigators in clinical Gerry Graham (University of Glasgow) will academic urology. investigate how innate immune responses to oncolytic adenovirus and HSV modulate overall Leukaemias are a major disease-specific anti-cancer efficacy. The relationship between research interest in the Institute of Cancer p53 mutation, inflammation and survival in Sciences, led by Tessa Holyoake (Director, high-grade serous ovarian cancer is being POG-LRC). These diseases of the haemopoietic The Autophagy group (Vignir Helgason) is lymphocyte lineage commitment and pursued by Patricia Roxburgh (Clinical Lecturer system arise from normal stem and progenitor focused on the inhibition of autophagy, a development. While investigating the role of in Medical Oncology) in collaboration with cells that have acquired corrupted cell fate survival process that can protect CML LSCs PKCα in early lymphocyte development, the Karen Vousden and Iain McNeish. decisions. These aberrant leukaemic stem cells following tyrosine kinase inhibition (TKI) drug group established that subversion of PKCα (LSCs) initiate and drive the process of treatment. The group has identified alternative signalling acts as an oncogenic trigger in B Breast cancer is the research focus for Iain leukaemogenesis. The research programmes signalling pathways that provide survival signals lymphocytes, resulting in the development of MacPherson, Torsten Stein and Joanne within the POG-LRC collectively focus on following oncoprotein inhibition, providing a chronic lymphocytic leukaemia. Edwards. Iain Macpherson studies the role understanding the fundamental mechanisms rationale for testing the effect of specific endo/exocytic trafficking in breast cancer in governing normal haemopoietic stem cell and inhibitors against these pathways on the survival The Stem Cell group (Helen Wheadon) focuses collaboration with Jim Norman’s group, LSC functions, identifying novel therapeutic of TKI-resistant cells and primary cells from on developmental pathways involved in normal ultimately with the aim of developing novel targets against LSCs and exploiting these targets TKI-resistant patients. and aberrant haemopoiesis, especially signals classes of therapeutic agents. A specific focus is in clinical trials. acting along conserved embryonic on the diacylglycerol kinase/Rab-coupling The Paediatric and Adult Acute Leukaemia morphogenic pathways. Increasing evidence protein axis that the Norman lab has previously The Chronic Myeloid Leukaemia (CML) group group (Karen Keeshan) is primarily focused on suggests that these embryonic morphogenic identified as a key regulator of integrin and (Tessa Holyoake) focuses on the identification mouse models and acute leukaemias. The pathways are important for controlling self- receptor tyrosine kinase trafficking from and characterisation of normal versus LSCs in group’s interest is in identifying and renewal behaviour of haemopoietic and cancer recycling endosomes, and to test the hypothesis CML aiming to elucidate survival factors or characterising novel factors that play a role in stem cells (CSCs). The main focus is to that this a major determinant of the pathways that selectively maintain leukaemic paediatric and adult leukaemia, extending into determine the role these pathways play in CSC aggressiveness of luminal B and HER2-positive stem and progenitor cells, and to develop novel the study of molecular events that have a role in self-renewal and leukaemia disease breast cancers. Torsten Stein’s interest is in approaches to target LSC maintenance. A stem and progenitor cell function and lineage progression. mammary gland morphogenesis and how systems biology approach to compare normal fate in normal haematopoiesis and in both adult these processes may be hijacked or deregulated versus LSCs in terms of gene, miRNA and and paediatric AML. The Leukaemia Stem Cell Self-Renewal group in breast cancer. He has demonstrated that protein expression and activity, and to (Mhairi Copland) studies the Notch and annexin A8 expression is associated with a understand drug resistance in the LSC The Molecular Lymphopoiesis group (Alison Hedgehog signalling pathways in myeloid subgroup of breast cancers with poor population, is being adopted. Michie) initially focused research efforts on leukaemias, and quiescence as a mechanism of prognosis, including hereditary breast cancers investigating the molecular events that regulate drug resistance in LSCs. Additional research

66 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE INSTITUTE OF CANCER SCIENCES 67 INSTITUTE OF CANCER SCIENCES (CONTINUED)

interests include the interactions between LSCs Translational research often requires high standards that allow us to make important consolidated our longstanding collaborative and their bone marrow niche, in particular via quality human tissue samples, which are decisions on the drug development pathway links with our research active cancer researchers the CXCL12/CXCR4 axis, and validation of collected with full patient consent and based on the results of these bioassays. These in NHS Greater Glasgow & Clyde, particularly induced pluripotent stem cells as an acceptable accompanying clinical, pathological and include PK studies in support of through the NHS Research Scotland research alternative model for preclinical drug screening follow-up information. In Glasgow, such tissue hydroxychloroquine (CML) and docetaxel awards from the Chief Scientist Office, Scotland. in haematological malignancies. samples are collected, stored and distributed to (prostate cancer), and PD assays of Mdm2 researchers by the Glasgow Bio-Repository, led (prostate cancer) and PARP inhibition Public engagement remains an important The Experimental Radiation Oncology group by James Going. This resource is funded by the (pancreatic cancer) and investigation of the role theme to our research programmes and Chloe (Rob Mairs) is developing targeted strategies NHS, Chief Scientist Office and CRUK (via the of markers associated with cellular senescence Cowan, CRUK Senior Research and Information to improve the biological specificity and Glasgow ECMC), and its importance to research in blood samples from patients with gastro- Nurse, leads on public engagement and effectiveness of radiation treatment by the has been underscored by the successful oesophageal cancer during chemotherapy education. In addition to our research selective delivery of radionuclides to tumour contribution to phase 1 of CRUK’s Stratified treatment. Exploratory studies of novel programmes, education remains an important cells, by enhanced uptake of radiolabelled Medicine Programme, led by Karin Oien. The biomarkers include collaborations with Gerry core activity and Karin Oien, Katherine West and targeting agents and by the selective concept of molecular profiling to optimise Graham (University of Glasgow, melanoma), Torsten Stein have led the development of new sensitisation of tumour cells to radiation. patient management is currently being explored Chiara Braconi (HCC), and Rhian Touyz postgraduate taught courses that will Their observations of synergy between in the national stratified medicine trial in patients (University of Glasgow) into mechanisms of commence in 2014. targeted therapy using [131I]meta- with advanced lung cancer and in the CUP-ONE hypertension induced by VEGF inhibition. iodobenzylguanidineand topotecan without study, which evaluates a number of molecular marrow toxicity provided the rationale for classifiers in patients with carcinoma of The CRUK Clinical Trials Unit (Rob Jones) current combination approaches applied unknown primary to refine treatment regimens develops, co-ordinates and delivers national clinically in Europe and the USA. based on the molecular classification of the and international multicentre studies, and has tumour and its likely primary origin (Karin Oien). UKCRC (UK Clinical Research Collaboration) The key objectives of the Translational Radiation registration. Several new trials have been Biology group (Anthony Chalmers) are to The Glasgow Experimental Cancer Medicine designed and funded from a variety of sources evaluate the clinical potential of novel inhibitors Centre (ECMC), led by Jeff Evans, and the CRUK over the past year that will ensure our central of the DNA damage response when delivered in Glasgow Clinical Trials Unit are the conduits to place in the organisation of large-scale trials in combination with radiotherapy and/or translate our findings through to clinical the UK and internationally, and our chemotherapy. The effects of DNA repair evaluation, while Stefano Schipani leads the collaborations with industry via NCRN/CRUK inhibitors in combination with radiotherapy and Clinical Radiotherapy Research group in and the ECMC Combination Alliances continue chemotherapy have been evaluated using a functional imaging, stereotactic sblative to flourish. These trials play on our core panel of intracranial models of glioblastoma radiotherapy and drug-radiotherapy strengths in gynaecological cancer, GI cancer, that recapitulate the key features of the disease combination studies. urological malignancies and radiotherapy in patients. The mechanisms underlying the in studies, and we are also developing a portfolio vivo effects are being investigated in two- and The objectives of the Glasgow ECMC are to take of studies in lung cancer. Many of these studies three-dimensional in vitro models of ideas from laboratory programmes into clinical reflect our growing expertise in collaborating glioblastoma that incorporate glioma stem cells, studies, with a specific emphasis of early phase with international collaborative groups, hypoxia and other features of the tumour clinical trials (including industry alliances) and in including the IRCI (International Rare Cancer micro-environment. The effects of novel biological markers including pharmacokinetic, Initiative). Specific highlights are the completion anti-invasive drugs on glioblastoma models in predictive and pharmacodynamic bioassays. of accrual to the SCOT study, which recruited vitro and in vivo are being pursued in Highlights of the early phase trials programme 6,087 patients from 237 investigator sites across collaboration with Mike Olson and Martin include the completion of the phase 1b 7 countries, and completion of accrual to KPS Drysdale, supported by the Brain Tumour component of an ECMC- Astra Zeneca Alliance (Ketamine Pain Study), which is the largest Charity. The clinical potential of these study (FGFR inhibitor) and a successful funding randomised phase 3 trial of pain intervention in approaches are being evaluated in a number of application for an Alliance study to support a the UK (n=214). In support of the clinical trials early phase clinical studies of PARP inhibition in chemo-radiation study in locally advanced strategy, Rob Jones, Iain McNeish, Iain combination with chemotherapy in recurrent pancreatic cancer. The laboratory component Macpherson, Anthony Chalmers, Andrew glioblastoma and with radiotherapy in newly of the ECMC is the Analytical Services Unit (ASU) Biankin, Karin Oien, Mhairi Copland and Jeff diagnosed disease. in which sample acquisition, handling, Evans are all members of NCRN Clinical Studies processing and reporting are all performed to Groups or sub-groups, and we have

68 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE INSTITUTE OF CANCER SCIENCES 69 RESEARCH SUPPORT AND MANAGEMENT

CANCER RESEARCH UK BEATSON INSTITUTE

Research Facilities Publications Conferences and Workshops Seminars Studentships and Postdoctoral Fellowships Administration Thanks for Supporting Us Patrons and Board of Governors Contact Details

71 The Histology Service performs essential A comprehensive immunohistochemistry RESEARCH FACILITIES processing of tissue samples and cellular service is offered using our two large capacity material from the wide range of cancer models autostainers. We are continually expanding the developed within the Institute, allowing the number of optimised antibodies that, if required, material to be evaluated at a cellular level in can be batch-stained using an autostainer to order to understand the disease mechanics. provide high quality, consistent staining. New The Service offers processing for tissue samples antibodies can also be optimised to produce a fixed in an array of different types of fixative working protocol that allows the antibody to be dependent on required subsequent analysis. used either on an autostainer or for hand Once received the tissue samples will be staining by the researcher. Training can be trimmed, appropriately processed and then provided in order that an individual scientist can orientated into paraffin wax blocks to facilitate understand the rationale and techniques Research Facilities supports research groups at the Beatson tissue sectioning and staining. The tissue available to allow them to perform the staining samples are processed according to type and to an acceptable standard. Institute and University of Glasgow on the Beatson site. This year necessity using previously designated specific, we said farewell to Robert McFarlane who retired as Laboratory specialised processing cycles. We have three The Institute has a Leica LMD6500 laser large capacity automated tissue processors microdissection system that allows Manager after 42 years at the Beatson. We welcomed Laura allowing large-scale consistent processing but subpopulations of tissue cells to be procured Bence who joined in May and took over as the new Laboratory when required specialised processing cycles from histological prepared slides under can be designed. Other material such as microscopic visualisation. We are able to cut Manager. We congratulate Richard Selkirk our Health and Safety organotypic assays, cell pellets, spheroids and sections from both cryostat and paraffin blocks Officer who has attained chartered membership of the Institute agar plugs can also be processed to provide a onto specialised slides, which can be stained Head of Research Facilities of Occupational Health and Safety (IOSH). There has been wax block to allow sectioning and further appropriately allowing cellular material to be Sue Fowler investigation. All paraffin wax blocks sectioned identified and separated to permit subsequent continued investment in new equipment for Information are stained with haematoxylin and eosin in order downstream analysis to be performed. to allow general analysis of cell morphology Consultation regarding the downstream Services and Histology. Information Services have invested in and structure. After initial analysis more analysis is imperative prior to work beginning as new equipment to increase the available network storage and specialised histology stains can be performed if this allows the correct protocols and procedures required to investigate specific tissue structures. to be used to maximise the results obtained Histology has purchased a fully automated Leica slide scanner from the specific analysis required. Both DNA for the capture and storage of high quality digital images. Where fixation is not required or and RNA material can be retrieved from the disadvantageous to tissue structure and tissue sections for downstream analysis. Building Facilities have been active with a number of projects to analysis, the facility offers a frozen section adapt laboratory space to accommodate specialist equipment. resource. Frozen tissue, embryos or cells A fully automated large capacity Leica SCN400F can be sectioned and when required slide scanner has been installed into Histology stained for examination using routine capable of capturing bright-field or fluorescent Building Facilities Central Services immunohistochemical or immunofluoresence images. This allows high quality digital images to Alistair Wilson, Alex Kernahan, Michael Daly, Margaret Laing (Supervisor), Elizabeth staining methods. Material for PCR analysis and be scanned and stored and if required Don MacBean1 Cheetham, Barbara Donnelly, Barbara Lambie, immunofluoresence investigation can also be quantitative interpretation can be performed. 1 until January Fiona McKay, Kirstie McPherson, Tracy Shields, sectioned from both paraffin-embedded Rose Steel, Robert Storey material and frozen tissue. Building Facilities manage the outsourced services provision for catering, cleaning and Central Services perform a wide range of duties janitorial services. We provide maintenance that are essential for the support of the research support for the Beatson Institute buildings and groups across the site. This includes cleaning manage alterations and refurbishments. This and sterilisation of reusable laboratory year the workshop repair and modification glassware, sterilisation of consumables, and service was closed due to space constraints, as preparation of tissue culture solutions, bacterial the building where it was located is due for culture media and Drosophila food. The team is demolition. also responsible for cleaning and checking items such as centrifuge rotors, X-ray Use of the online helpdesk facility continues to processors, water baths and pH meters. The be an effective means of logging reactive calls stocking of tissue culture suites, laboratory for maintenance and repair. Minor project work waste collections and autoclave processing to continues at a fairly high level. A number of make waste safe are performed daily. building services have been altered or extended to accommodate new equipment. This year PAT Histology Service testing has been completed for all portable Colin Nixon, Saira Ghafoor, Mark Hughes, appliances in the Institute. Wendy Lambie, Fiona McGregor, Brenda McGuire, Vivienne Morrison

72 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH FACILITIES 73 Information Services running large data analyses, in some outgoing orders to ensure compliance with Peter McHardy, Iain White cases reducing data analysis runs from days Institute safety procedures, particularly those to minutes. relating to COSHH. Information Services provide a wide range of support services, including server support, A range of replacement hardware is stored on Service contracts for core equipment are hardware cover, an on-site helpdesk providing site to allow fast repairs. A good selection of procured centrally and maintenance or repairs both repair and software support as well as help loan IT hardware, from USB drives to digital are coordinated to ensure these are dealt with as in hardware selection and user training. There projectors, is held centrally. We provide video efficiently as possible. This year we have are over 350 users with nearly 400 PCs on site conferencing facilities, enabling conference installed a new freezer alarm system that allows comprising a mixture of Windows computers, calls between the Beatson and other Cancer us to monitor and interrogate freezer alarms Apple Macs and Linux machines, with central Research UK sites as well as many other remotely. This enables us to attend to alarm authentication, central file store and print locations. Audio-visual support services for situations more efficiently and to forestall any sharing. large conferences have been provided at a potential issues before a freezer fails. number of international venues, as well as The servers provide in excess of 500 TB of overseeing the in-house 178-seat state-of-the- The stores facility stocks a wide range of online storage with nightly backups and tapes art lecture theatre. consumables with rapid re-stocking to ensure stored off-site, to provide support for high use materials are always available. Items New Laboratory Manager microscopy, DNA sequencing and mass can be withdrawn on a self-service basis with Laura Bence spectrometry data. Data backup facilities are automatic cost centre allocation via swipe card. now available for laptop users when they are A porter service is run to deliver external orders off-site, aimed at reducing data loss. to the researchers, while stores processes any outgoing samples or materials for courier Human cell line authentication using the All PCs are built with a common desktop collection. We continue to review the services Promega Geneprint 10 Kit is available as an environment, around Windows or Mac OS X and provided by stores to try to improve what is on internal service. The samples are run on the Microsoft Office and are actively managed and offer to the scientific staff. This has required Applied Biosystems 3130xl Sequencer (Gene upgraded to ensure the best possible working negotiating preferential pricing with our Fragment Analysis) and analysed using environment. Mac OS X Mountain Lion has been suppliers at a local level. As a result of these Genemapper v4.0 software (Applied rolled out across the site and we have negotiations and better turnaround times from Biosystems). Regular cell line authentication is completed the upgrade of relevant Windows suppliers, we have been able to reduce the important both to confirm integrity of data and computers to Windows 7. All e-mail services run overall value of stock held without is increasingly requested by journals as a on Microsoft Exchange which allows local compromising supply lines to the laboratories. requirement prior to publication client-based access and web access to email as well as delivering email, diaries and address Reagent Services provide a diverse range of books to mobile devices including iPhones, Molecular Technology and Reagent Services support to the research groups. The iPads and other smart phones. Laboratory Management Billy Clark, Deborah Gardner, Andrew Keith mycoplasma screening service offers testing of Laura Bence1, Robert McFarlane2, Richard each research group’s cells every four to five We continue to migrate over as many physical Selkirk, Michael McTaggart, Joe McFadden, The Molecular Technology Service provides months. Researchers are also encouraged to servers as possible to virtual servers using George Monteith routine plasmid sequencing and DNA have newly imported cell lines tested as soon as VMware. We provide access to virtualised 1 from May, 2 until August purification on a small and large-scale. possible after arrival as we have found that a servers for research groups allowing them Sequencing is performed on an Applied significant number of newly imported cell lines greater flexibility for test and production Laboratory Management is responsible for Biosystems 3130xl (16 capillary) Sequencer that are infected with mycoplasma. Cell lines are applications. This also allows us to provide providing advice and information to scientists provides good sample throughput, long read mainly tested using a luciferase assay that virtual workstations for researchers with both on health and safety, particularly on how to lengths and a sample turnaround time of 24 detects mycoplasmal enzymes. They may also high core counts and large amounts of RAM, carry out risk assessments and on appropriate hours. In recent years, DNA sequencing has be tested by: Hoechst staining to detect the making them ideal for mass spectroscopy control measures. Safety plays an important part been revolutionised by the introduction of next presence of mycoplasma DNA; enzyme analysis or other computationally intense of everyday life in the laboratory and in running generation technologies offering large-scale immunoassay against the four most common applications. the building services. We administer the sequencing in a matter of days. This year, we species of mycoplasma; or colorimetric monitoring and training elements on a have sequenced a number of genomic libraries microplate assay to detect 16S ribosomal Significant investment has been put into day-to-day basis, identifying training needs and on our Illumina GAIIx sequencer applying mycoplasma RNA. providing documentation and procedures to ensuring adequate provision is made to fulfil the ChIP-seq and RNA-seq protocols. Multiplexing allow us to run the service in a manner Institute’s legal obligations to staff. This year we has enabled us to sequence more than one Cell-derived matrices from Tiff 5 cells are commensurate with ITIL. This has been used as have trained a number of new first aiders and library per lane, increasing throughput while prepared to order for the research groups and the foundation of our business continuity fire marshalls. To enhance safety we have reducing time and costs. Recently we have have proved very popular. Stocks of commonly documentation and has led to the revision and introduced an improved training programme for taken over the library preparation from the used tissue culture medium are ordered and the continual improvement of many of our users of our cryostore and radiation suites. research groups. batch testing of serum is coordinated. The day-to-day working practices. facility provides a range of commonly used A major function of Laboratory Management is Small-scale DNA purification is performed on a buffers, for example 10X TBST and bacterial Our intranet uses a content management the overseeing of shared equipment servicing, Qiagen 8000 Biorobot. Researchers provide growth reagents. Each product is tested for system (CMS) framework, allowing service replacement and the purchase of new overnight bacterial cultures that are processed suitability of use and sterility where possible managers and support departments the ability equipment to facilitate the needs of researchers. by the facility. Sample numbers are consistently before being released for general stock. The to easily upload forms and information for users. We have effective procurement processes and in the region of 15,000-17,000 per year. We preparation of antibiotic bacterial culture plates Bespoke hardware systems have been designed liaise with Cancer Research UK purchasing to continue to provide a very popular large-scale is automated using the Mediaclave (Integra and configured for users allowing them to take advantage of any centralised agreements. A DNA purification (maxiprep) service from Biosciences AG) to sterilise and dispense into achieve significant speed gains when they are further essential role is the monitoring of all bacterial cultures. the plates.

74 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH FACILITIES 75 RESEARCH PUBLICATIONS

Kurt Anderson (page 24) von Thun A, Preisinger C, Rath O, Schwarz JP, ROS production and NF-kappaB activation Jeff Evans (page 26) Tumour Cell Migration Ward C, Monsefi N, Rodriguez J, Garcia-Munoz A, triggered by RAC1 facilitate WNT-driven intestinal Translational Cancer Therapeutics Birtwistle M, Bienvenut W, Anderson KI, Kolch W, stem cell proliferation and colorectal cancer von Kriegsheim A. initiation. Cell Stem Cell 2013; 12: 761-73 Primary Research Papers Extracellular Signal-Regulated Kinase Regulates Primary Research Papers Müllenbroich CM, McGhee EJ, Wright AJ, Acosta JC, Banito A, Wuestefeld T, Georgilis A, RhoA Activation and Tumor Cell Plasticity by Pawlikowski JS, McBryan T, van Tuyn J, Drotar Anderson KI, Mathieson K. Janich P, Morton JP, Athineos D, Kang TW, Inhibiting Guanine Exchange Factor H1 Activity. Mol ME, Hewitt RN, Maier AB, King A, Blyth K, Wu H, Adaptive nonlinear microscopy for whole tissue Lasitschka F, Andrulis M, Pascual G, Morris KJ, Khan Cell Biol 2013; 33: 4526-37 Adams PD. imaging. , Multiphoton Microscopy S, Jin H, Dharmalingam G, Snijders AP, Carroll T, Proc. SPIE 8588 Wnt signaling potentiates nevogenesis. Proc Natl in the Biomedical Sciences XIII, 85881X (February Capper D, Pritchard C, Inman GJ, Longerich T, Other Publications Acad Sci USA 2013; 110: 16009-14 22, 2013); doi:10.1117/12.2006375 Sansom OJ, Benitah SA, Zender L, Gil J. Nobis M, Carragher NO, McGhee EJ, Morton JP, A complex secretory program orchestrated by the Sansom OJ, Anderson KI, Timpson P. Radulescu S, Ridgway RA, Cordero J, Athineos D, Müllenbroich CM, McGhee EJ, Wright AJ, inflammasome controls paracrine senescence. Advanced intravital subcellular imaging reveals vital Salgueiro P, Poulsom R, Neumann J, Jung A, Patel Nat Anderson KI, Mathieson K. 2013; 15: 978-90 three-dimensional signalling events driving cancer S, Woodgett J, Barker N, Pritchard DM, Oien K, Cell Biol Strategies to overcome photobleaching in cell behaviour and drug responses in live tissue. Sansom OJ. Acute WNT signalling activation algorithm-based adaptive optics for nonlinear Denley SM, Jamieson NB, McCall P, Oien KA, FEBS J 2013; 280: 5177-97 perturbs differentiation within the adult stomach in-vivo imaging. . 2014; 19: 16021 Morton JP, Carter CR, Edwards J, McKay CJ. J Biomed Opt and rapidly leads to tumour formation. Oncogene Activation of the IL-6R/Jak/stat pathway is 2013; 32: 2048-57 Myant KB, Cammareri P, McGhee EJ, Ridgway RA, associated with a poor outcome in resected Huels DJ, Cordero JB, Schwitalla S, Kalna G, Ogg Karen Blyth (page 53) pancreatic ductal adenocarcinoma. Wiese KE, Walz S, von Eyss B, Wolf E, J Gastrointest EL, Athineos D, Timpson P, Vidal M, Murray GI, Transgenic Models of Cancer 2013; 17: 887-98 Athineos D, Sansom O, Eilers M. Surg Greten FR, Anderson KI, Sansom OJ. The Role of MIZ-1 in MYC-Dependent ROS production and NF-kappaB activation Primary Research Papers Karim SA, Creedon H, Patel H, Carragher NO, Tumorigenesis. Cold Spring Harb Perspect Med triggered by RAC1 facilitate WNT-driven intestinal Acosta JC, Banito A, Wuestefeld T, Georgilis A, Morton JP, Muller WJ, Evans TR, Gusterson B, 2013; 3: a014290 stem cell proliferation and colorectal cancer Janich P, Morton JP, Athineos D, Kang TW, Sansom OJ, Brunton VG.

initiation. 2013; 12: 761-73 Lasitschka F, Andrulis M, Pascual G, Morris KJ, Khan Dasatinib inhibits mammary tumour development Cell Stem Cell Zheng L, Mackenzie ED, Karim SA, Hedley A, S, Jin H, Dharmalingam G, Snijders AP, Carroll T, in a genetically engineered mouse model. Blyth K, Kalna G, Watson DG, Szlosarek P, J Pathol Nobis M, McGhee EJ, Morton JP, Schwarz JP, Capper D, Pritchard C, Inman GJ, Longerich T, 2013; 230: 430-40 Frezza C, Gottlieb E. Karim SA, Quinn J, Edward M, Campbell AD, Sansom OJ, Benitah SA, Zender L, Gil J. Reversed argininosuccinate lyase activity in McGarry LC, Evans TR, Brunton VG, Frame MC, A complex secretory program orchestrated by the Lassen U, Miller WH, Hotte S, Evans TR, fumarate hydratase-deficient cancer cells. Carragher NO, Wang Y, Sansom OJ, Timpson P, inflammasome controls paracrine senescence. Kollmansberger C, Adamson D, Nielsen DL, Spicer Nat Cancer Metab 2013; 1: 12 Anderson KI. Cell Biol 2013; 15: 978-90 J, Chen E, Meyer T, Brown K, Rafi R, Sawyer MB. Intravital FLIM-FRET imaging reveals dasatinib- Phase I evaluation of the effects of ketoconazole induced spatial control of src in pancreatic cancer. Cheung EC, Athineos D, Lee P, Ridgway RA, and rifampicin on cediranib pharmacokinetics in Cancer Res 2013; 73: 4674-86 Lambie W, Nixon C, Strathdee D, Blyth K, Sansom Martin Drysdale (page 44) patients with solid tumours. Cancer Chemother OJ, Vousden KH. Drug Discovery Programme Pharmacol 2013; 71: 543-9 Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, TIGAR is required for efficient intestinal Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, regeneration and tumorigenesis. Dev Cell Other publications Macpherson IR, Poondru S, Simon GR, Gedrich Ridgway R, Adams PD, Anderson KI, Gottlieb E, 2013; 25: 463-77 R, Brock K, Hopkins CA, Stewart K, Stephens A, Drysdale MJ. Sansom OJ, Ryan KM. Evans TR. Fragment-based Drug Discovery: the Shape of p53 status determines the role of autophagy in Hock AK, Lee P, Maddocks OD, Mason SM, Blyth K, A phase 1 study of OSI-930 in combination with Things to Come. Aust J Chem 2013; 66: 1544-9 pancreatic tumour development. Nature Vousden KH. erlotinib in patients with advanced solid tumours. 2013; 504: 296-300 iRFP is a sensitive marker for cell number and tumor Eur J Cancer 2013; 49: 782-9 Morley AD, Pugliese A, Birchall K, Bower J, Brennan growth in high-throughput systems. Cell Cycle P, Brown N, Chapman T, Drysdale M, Gilbert IH, Trinidad AG, Muller PA, Cuellar J, Klejnot M, Nobis 2013; 13: 220-6 Nobis M, Carragher NO, McGhee EJ, Morton JP, Hoelder S, Jordan A, Ley SV, Merritt A, Miller D, M, Valpuesta JM, Vousden KH. Sansom OJ, Anderson KI, Timpson P. Swarbrick ME, Wyatt PG. Interaction of p53 with the CCT complex promotes Myant KB, Cammareri P, McGhee EJ, Ridgway RA, Advanced intravital subcellular imaging reveals vital Fragment-based hit identification: thinking in 3D. protein folding and wild-type p53 activity. Mol Cell Huels DJ, Cordero JB, Schwitalla S, Kalna G, Ogg three-dimensional signalling events driving cancer Drug Discov Today 2013; 18: 1221-7 2013; 50: 805-17 EL, Athineos D, Timpson P, Vidal M, Murray GI, cell behaviour and drug responses in live tissue. Greten FR, Anderson KI, Sansom OJ. FEBS J 2013; 280: 5177-97

76 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 77 Nobis M, McGhee EJ, Morton JP, Schwarz JP, Steele CW, Jamieson NB, Evans TR, McKay CJ, Zheng L, Mackenzie ED, Karim SA, Hedley A, Itakura A, Aslan JE, Kusanto BT, Phillips KG, Porter Karim SA, Quinn J, Edward M, Campbell AD, Sansom OJ, Morton JP, Carter CR. Blyth K, Kalna G, Watson DG, Szlosarek P, Frezza C, JE, Newton PK, Nan X, Insall RH, Chernoff J, McGarry LC, Evans TR, Brunton VG, Frame MC, Exploiting inflammation for therapeutic gain in Gottlieb E. McCarty OJ. Carragher NO, Wang Y, Sansom OJ, Timpson P, pancreatic cancer. Br J Cancer 2013; 108: 997-1003 Reversed argininosuccinate lyase activity in p21-Activated kinase (PAK) regulates cytoskeletal Anderson KI. fumarate hydratase-deficient cancer cells. reorganization and directional migration in human Intravital FLIM-FRET imaging reveals dasatinib- Cancer Metab 2013; 1: 12 neutrophils. PLoS One 2013; 8: e73063 induced spatial control of src in pancreatic cancer. Eyal Gottlieb (page 12) Cancer Res 2013; 73: 4674-86 Apoptosis and Tumour Metabolism King JS, Gueho A, Hagedorn M, Gopaldass N, Danny Huang (page 14) Leuba F, Soldati T, Insall RH. Poplin E, Wasan H, Rolfe L, Raponi M, Ikdahl T, WASH is required for lysosomal recycling and Primary Research Papers Ubiquitin Signalling Bondarenko I, Davidenko I, Bondar V, Garin A, efficient autophagic and phagocytic digestion. Gonzalvez F, D’Aurelio M, Boutant M, Moustapha A, Boeck S, Ormanns S, Heinemann V, Bassi C, Evans Mol Biol Cell 2013; 24: 2714-26 Puech JP, Landes T, Arnaune-Pelloquin L, Vial G, Primary Research Papers TR, Andersson R, Hahn H, Picozzi V, Dicker A, Taleux N, Slomianny C, Wanders RJ, Houtkooper Dou H, Buetow L, Sibbet GJ, Cameron K, Mann E, Voong C, Kaur P, Isaacson J, Allen A. Park L, Thomason PA, Zech T, King JS, Veltman RH, Bellenguer P, Moller IM, Gottlieb E, Vaz FM, Huang DT. Randomized, Multicenter, Phase II Study of CO-101 DM, Carnell M, Ura S, Machesky LM, Insall RH. Manfredi G, Petit PX. Essentiality of a non-RING element in priming Versus Gemcitabine in Patients With Metastatic Cyclical action of the WASH complex: FAM21 and Barth syndrome: cellular compensation of donor ubiquitin for catalysis by a monomeric E3. Pancreatic Ductal Adenocarcinoma: Including a capping protein drive WASH recycling, not initial mitochondrial dysfunction and apoptosis inhibition 2013; 20: 982-6 Prospective Evaluation of the Role of hENT1 in Nat Struct Mol Biol recruitment. Dev Cell 2013; 24: 169-81 due to changes in cardiolipin remodeling linked to Gemcitabine or CO-101 Sensitivity. J Clin Oncol tafazzin (TAZ) gene mutation. 2013; 31: 4453-61 Biochim Biophys Sapey E, Greenwood H, Walton G, Mann E, Love A, 2013; 1832: 1194-206 Acta Robert Insall (page 28) Aaronson N, Insall RH, Stockley RA, Lord JM. Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, Cell Migration and Chemotaxis Phosphoinositide 3 kinase inhibition restores Jalmar O, Francois-Moutal L, Garcia-Saez AJ, Perry Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, neutrophil accuracy in the elderly: towards targeted M, Granjon T, Gonzalvez F, Gottlieb E, Ayala- Ridgway R, Adams PD, Anderson KI, Gottlieb E, Primary Research Papers treatments for immunesenescence. Blood 2014 Sanmartin J, Klosgen B, Schwille P, Petit PX. Sansom OJ, Ryan KM. Davidson AJ, King JS, Insall RH. 2014; 123: 239-48 Epub 2013/11/06 Caspase-8 binding to cardiolipin in giant p53 status determines the role of autophagy in The use of streptavidin conjugates as immunoblot unilamellar vesicles provides a functional docking pancreatic tumour development. Nature 2013; 504: loading controls and mitochondrial markers for use Other Publications platform for bid. PLoS One 2013; 8: e55250 296-300 with Dictyostelium discoideum. Biotechniques Insall R. 2013; 55: 39-41 The interaction between pseudopods and Kaplon J, Zheng L, Meissl K, Chaneton B, Selivanov Tan EH, Morton JP, Timpson P, Tucci P, Melino G, extracellular signalling during chemotaxis and VA, Mackay G, van der Burg SH, Verdegaal EM, Flores ER, Sansom OJ, Vousden KH, Muller PA. Davidson AJ, Ura S, Thomason PA, Kalna G, directed migration. Curr Opin Cell Biol 2013; 25: Cascante M, Shlomi T, Gottlieb E*, Peeper DS*. Functions of TAp63 and p53 in restraining the Insall RH. 526-31 A key role for mitochondrial gatekeeper pyruvate development of metastatic cancer. Oncogene, Abi Is Required for Modulation and Stability but Not dehydrogenase in oncogene-induced senescence. published online 22 Jul 2013, doi: 10.1038/ Localization or Activation of the SCAR/WAVE Insall RH. Nature 2013; 498: 109-12 onc.2013.287 Complex. Eukaryot Cell 2013; 12: 1509-16 WASH complex conservation: don’t WASH away *corresponding authors the family! Trends Cell Biol 2013; 23: 519-20 Venugopal B, Baird R, Kristeleit RS, Plummer R, Cowan R, Stewart A, Fourneau N, Hellemans P, Long JS, Crighton D, O’Prey J, Mackay G, Zheng L, Elsayed Y, McClue S, Smit JW, Forslund A, Phelps C, Palmer TM, Gottlieb E, Ryan KM. Camm J, Evans TR, de Bono JS, Banerji U. Extracellular adenosine sensing-a metabolic cell A phase I study of quisinostat (JNJ-26481585), an death priming mechanism downstream of p53. Mol oral hydroxamate histone deacetylase inhibitor with Cell 2013; 50: 394-406 evidence of target modulation and antitumor activity, in patients with advanced solid tumors. Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, Clin Cancer Res 2013; 19: 4262-72 Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, Ridgway R, Adams PD, Anderson KI, Gottlieb E, Xu J, Evans TJ, Coon C, Copley-Merriman K, Su Y. Sansom OJ, Ryan KM. Measuring patient-reported outcomes in advanced p53 status determines the role of autophagy in gastric cancer. Ecancermedicalscience 2013; 7: 351 pancreatic tumour development. Nature 2013; 504: 296-300 Zheng L, Mackenzie ED, Karim SA, Hedley A, Blyth K, Kalna G, Watson DG, Szlosarek P, Frezza C, Tannahill GM, Curtis AM, Adamik J, Palsson- Gottlieb E. McDermott EM, McGettrick AF, Goel G, Frezza C, Reversed argininosuccinate lyase activity in Bernard NJ, Kelly B, Foley NH, Zheng L, Gardet A, fumarate hydratase-deficient cancer cells. Tong Z, Jany SS, Corr SC, Haneklaus M, Caffrey BE, Cancer Metab 2013; 1: 12 Pierce K, Walmsley S, Beasley FC, Cummins E, Nizet V, Whyte M, Taylor CT, Lin H, Masters SL, Other Publications Gottlieb E, Kelly VP, Clish C, Auron PE, Xavier RJ, Morton JP, Sansom OJ. O’Neill LA. MYC-y mice: from tumour initiation to therapeutic Succinate is an inflammatory signal that induces targeting of endogenous MYC. Mol Oncol 2013; 7: IL-1beta through HIF-1alpha. Nature 2013; 496: 248-58 238-42

78 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 79 Davidson AJ, Ura S, Thomason PA, Kalna G, Forbat L, Place M, Hubbard G, Leung H, Kelly D. Laura Machesky (page 30) Insall RH. The role of interpersonal relationships in men’s Migration, Invasion and Metastasis Abi Is Required for Modulation and Stability but attendance in primary care: qualitative findings in a Not Localization or Activation of the SCAR/WAVE cohort of men with prostate cancer. Support Care Complex. Eukaryot Cell 2013; 12: 1509-16 Cancer 2014; 22: 409-15 Primary Research Papers Futo K, Bodis E, Machesky LM, Nyitrai M, Myant KB, Cammareri P, McGhee EJ, Ridgway RA, Neuhaus J, Schiffer E, von Wilcke P, Bauer HW, Visegrady B. Huels DJ, Cordero JB, Schwitalla S, Kalna G, Ogg Leung H, Siwy J, Ulrici W, Paasch U, Horn LC, Membrane binding properties of IRSp53-missing in EL, Athineos D, Timpson P, Vidal M, Murray GI, Stolzenburg JU. metastasis domain (IMD) protein. Biochim Biophys Greten FR, Anderson KI, Sansom OJ. Seminal plasma as a source of prostate cancer Acta 2013; 1831: 1651-5 ROS production and NF-kappaB activation peptide biomarker candidates for detection of triggered by RAC1 facilitate WNT-driven intestinal indolent and advanced disease. PLoS One 2013; 8: Ma Y, Li A, Faller WJ, Libertini S, Fiorito F, Gillespie stem cell proliferation and colorectal cancer e67514 DA, Sansom OJ, Yamashiro S, Machesky LM. initiation. Cell Stem Cell 2013; 12: 761-73 Fascin 1 is transiently expressed in mouse Patel R, Gao M, Ahmad I, Fleming J, Singh LB, Rai melanoblasts during development and promotes Zanivan S, Meves A, Behrendt K, Schoof EM, TS, McKie AB, Seywright M, Barnetson RJ, Edwards migration and proliferation. Development 2013; 140: Neilson LJ, Cox J, Tang HR, Kalna G, van Ree JH, J, Sansom OJ, Leung HY. 2203-11 van Deursen JM, Trempus CS, Machesky LM, Sprouty2, PTEN, and PP2A interact to regulate Linding R, Wickstrom SA, Fassler R, Mann M. prostate cancer progression. J Clin Invest 2013; 123: Ma Y, Reynolds LE, Li A, Stevenson RP, Hodivala- In vivo SILAC-based proteomics reveals 1157-75 Dilke KM, Yamashiro S, Machesky LM. phosphoproteome changes during mouse skin Fascin 1 is dispensable for developmental and carcinogenesis. Cell Rep 2013; 3: 552-66 Rajan P, Sudbery IM, Villasevil ME, Mui E, Fleming J, tumour angiogenesis. Biol Open 2013; 2: 1187-91 Davis M, Ahmad I, Edwards J, Sansom OJ, Sims D, Zheng L, Mackenzie ED, Karim SA, Hedley A, Blyth Ponting CP, Heger A, McMenemin RM, Pedley ID, Park L, Thomason PA, Zech T, King JS, Veltman K, Kalna G, Watson DG, Szlosarek P, Frezza C, Leung HY. DM, Carnell M, Ura S, Machesky LM, Insall RH. Muinonen-Martin AJ, Knecht DA, Veltman DM, Gottlieb E. Next-generation Sequencing of Advanced Prostate Cyclical action of the WASH complex: FAM21 and Thomason PA, Kalna G, Insall RH. Reversed argininosuccinate lyase activity in Cancer Treated with Androgen-deprivation capping protein drive WASH recycling, not initial Measuring chemotaxis using direct visualization fumarate hydratase-deficient cancer cells. Therapy. Eur Urol published online 14 Aug 2013, doi: recruitment. Dev Cell 2013; 24: 169-81 microscope chambers. Methods Mol Biol 2013; Cancer Metab 2013; 1: 12 10.1016/j.eururo.2013.08.011 1046: 307-21 Schachtner H, Calaminus SD, Sinclair A, Other Publications Shafique K, Proctor MJ, McMillan DC, Leung H, Monypenny J, Blundell MP, Leon C, Holyoake TL, Tyrrell BJ, Neilson M, Insall RH, Machesky LM. Muinonen-Martin AJ, Knecht DA, Veltman DM, Smith K, Sloan B, Morrison DS. Thrasher AJ, Michie AM, Vukovic M, Gachet C, Predicting cell shapes in melanomas. Pigment Cell Thomason PA, Kalna G, Insall RH. The modified Glasgow prognostic score in prostate Jones GE, Thomas SG, Watson SP, Machesky LM. Melanoma Res 2014; 27: 5-6 Measuring chemotaxis using direct visualization cancer: results from a retrospective clinical series of Megakaryocytes assemble podosomes that microscope chambers. Methods Mol Biol 2013; 744 patients. BMC Cancer 2013; 13: 292 degrade matrix and protrude through basement 1046: 307-21 membrane. Blood 2013; 121: 2542-52 Gabriela Kalna (page 49) Willder JM, Heng SJ, McCall P, Adams CE, Tannahill Bioinformatics C, Fyffe G, Seywright M, Horgan PG, Leung HY, Sonego M, Gajendra S, Parsons M, Ma Y, Hobbs Hing Leung (page 16) Underwood MA, Edwards J. C, Zentar MP, Williams G, Machesky LM, Doherty Androgen receptor phosphorylation at serine 515 P, Lalli G. Primary Research Papers Prostate Cancer Biology by Cdk1 predicts biochemical relapse in prostate Fascin regulates the migration of subventricular Ahmad I, Kalna G, Ismail M, Birrell F, Asterling S, cancer patients. Br J Cancer 2013; 108: 139-48 zone-derived neuroblasts in the postnatal brain. McCartney E, Greene D, Davies J, Leung HY. Primary Research Papers J Neurosci 2013; 33: 12171-85 Ahmad I, Gao M, Patel R, Leung HY. Prostate gland lengths and iceball dimensions Williamson SC, Hepburn AC, Wilson L, Coffey predict micturition functional outcome following Modelling synergistic interactions between HER2, K, Ryan-Munden CA, Pal D, Leung HY, Robson Tang H, Li A, Bi J, Veltman DM, Zech T, Spence salvage prostate cryotherapy in men with radiation Sprouty2 and PTEN in driving prostate CN, Heer R. HJ, Yu X, Timpson P, Insall RH, Frame MC, recurrent prostate cancer. PLoS One 2013; 8: e69243 carcinogenesis. Asian J Androl 2013; 15: 323-7 Characterisations of human prostate stem cells Machesky LM. reveal deficiency in class I UGT enzymes as a novel Loss of Scar/WAVE complex promotes Ahmad I, Singh LB, Yang ZH, Kalna G, Fleming J, Ahmad I, Kalna G, Ismail M, Birrell F, Asterling S, mechanism for castration-resistant prostate cancer. N-WASP- and FAK-dependent invasion. Curr Biol Fisher G, Cooper C, Cuzick J, Berney DM, Moller H, McCartney E, Greene D, Davies J, Leung HY. Br J Cancer 2013; 109: 950-6 2013 ; 23: 107-17 Scardino P, Leung HY. Prostate gland lengths and iceball dimensions Mir143 expression inversely correlates with nuclear predict micturition functional outcome following Other Publications Zanivan S, Meves A, Behrendt K, Schoof EM, ERK5 immunoreactivity in clinical prostate cancer. salvage prostate cryotherapy in men with radiation Zhang T, Watson DG, Wang L, Abbas M, Murdoch Neilson LJ, Cox J, Tang HR, Kalna G, van Ree JH, Br J Cancer 2013; 108: 149-54 recurrent prostate cancer. PLoS One 2013; 8: e69243 L, Bashford L, Ahmad I, Lam NY, Ng AC, Leung HY. van Deursen JM, Trempus CS, Machesky LM, Application of Holistic Liquid Chromatography- Linding R, Wickstrom SA, Fassler R, Mann M. Costea DE, Hills A, Osman AH, Thurlow J, Kalna G, Ahmad I, Singh LB, Yang ZH, Kalna G, Fleming J, High Resolution Mass Spectrometry Based Urinary In vivo SILAC-based proteomics reveals Huang X, Pena Murillo C, Parajuli H, Suliman S, Fisher G, Cooper C, Cuzick J, Berney DM, Moller H, Metabolomics for Prostate Cancer Detection and phosphoproteome changes during mouse skin Kulasekara KK, Johannessen AC, Partridge M. Scardino P, Leung HY. Biomarker Discovery. PLoS One 2013; 8: e65880 carcinogenesis. Cell Rep 2013; 3: 552-66 Identification of two distinct carcinoma-associated Mir143 expression inversely correlates with nuclear fibroblast subtypes with differential tumor- ERK5 immunoreactivity in clinical prostate cancer. promoting abilities in oral squamous cell Br J Cancer 2013; 108: 149-54 carcinoma. Cancer Res 2013; 73: 3888-901

80 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 81 Other Publications dissociation of Hsp90 from the ERK5-Cdc37 Ampe C, Machesky LM, Nyitrai M. complex. Mol Cell Biol 2013; 33: 1671-86 Biophysical approaches for investigation of the cytoskeleton. A special issue stemming from Gabrielsen M, Schuldt M, Munro J, Borucka D, research presented at the 2012 ECF meeting: A Cameron J, Baugh M, Mleczak A, Lilla S, Morrice N, FEBS/EMBO workshop lecture course in Pecs, Olson MF. Hungary. Cytoskeleton (Hoboken) 2013; 70: 539 Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an Li A, Machesky LM. exemplary target. Cell Commun Signal 2013; 11: 58 Rac1 cycling fast in melanoma with P29S. Pigment Cell Melanoma Res epublished 8 Feb 2013, doi: Gantke T, Boussouf S, Janzen J, Morrice NA, 10.1111/pcmr.12074 Howell S, Muhlberger E, Ley SC. Ebola virus VP35 induces high-level production of Schachtner H, Calaminus SD, Thomas SG, recombinant TPL-2-ABIN-2-NF-kappaB1 p105 Machesky LM. complex in co-transfected HEK-293 cells. Biochem Podosomes in adhesion, migration, J 2013; 452: 359-65 mechanosensing and matrix remodeling. Cytoskeleton (Hoboken) 2013; 70: 572-89 Prevost M, Chamousset D, Nasa I, Freele E, Morrice N, Moorhead G, Trinkle-Mulcahy L. Tyrrell BJ, Neilson M, Insall RH, Machesky LM. Quantitative fragmentome mapping reveals novel, Predicting cell shapes in melanomas. Pigment Cell domain-specific partners for the modular protein Melanoma Res 2014; 27: 5-6 RepoMan (recruits PP1 onto mitotic chromatin at anaphase). Mol Cell Proteomics 2013; 12: 1468-86

Gillian Mackay (page 50) Wickman GR, Julian L, Mardilovich K, Schumacher Metabolomics S, Munro J, Rath N, Zander SA, Mleczak A, Sumpton D, Morrice N, Bienvenut WV, Olson MF. matrix receptors are trafficked through the late Wickman GR, Julian L, Mardilovich K, Schumacher Blebs produced by actin-myosin contraction endosomal pathway in a way that dictates how cells S, Munro J, Rath N, Zander SA, Mleczak A, Primary Research Papers during apoptosis release damage-associated migrate. Bioessays 2013; 35: 523-32 Sumpton D, Morrice N, Bienvenut WV, Olson MF. Kaplon J, Zheng L, Meissl K, Chaneton B, Selivanov molecular pattern proteins before secondary Blebs produced by actin-myosin contraction VA, Mackay G, van der Burg SH, Verdegaal EM, necrosis occurs. Cell Death Differ 2013; 20: during apoptosis release damage-associated Cascante M, Shlomi T, Gottlieb E, Peeper DS. 1293-305 molecular pattern proteins before secondary A key role for mitochondrial gatekeeper pyruvate Michael Olson (page 34) necrosis occurs. Cell Death Differ 2013; 20: dehydrogenase in oncogene-induced senescence. Molecular Cell Biology 1293-305 Nature 2013; 498: 109-12 Jim Norman (page 32) Primary Research Papers Other Publications Long JS, Crighton D, O’Prey J, Mackay G, Zheng L, Integrin Cell Biology Gabrielsen M, Schuldt M, Munro J, Borucka D, Olson MF. Palmer TM, Gottlieb E, Ryan KM. Cameron J, Baugh M, Mleczak A, Lilla S, Morrice N, Finding the shape-shifter genes. Nat Cell Biol Extracellular adenosine sensing-a metabolic cell Primary Research Papers Olson MF. 2013; 15: 723-5 death priming mechanism downstream of p53. Mol Campbell AD, Lawn S, McGarry LC, Welch HC, Cucurbitacin covalent bonding to cysteine thiols: Cell 2013; 50: 394-406 Ozanne BW, Norman JC. the filamentous-actin severing protein Cofilin1 as an P-Rex1 cooperates with PDGFRbeta to drive cellular exemplary target. Cell Commun Signal 2013; 11: 58 Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, migration in 3D microenvironments. PLoS One Kevin Ryan (page 18) Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, 2013; 8: e53982 Ibbetson SJ, Pyne NT, Pollard AN, Olson MF, Tumour Cell Death Ridgway R, Adams PD, Anderson KI, Gottlieb E, Samuel MS. Sansom OJ, Ryan KM. Jacquemet G, Green DM, Bridgewater RE, von Mechanotransduction pathways promoting tumor Primary Research Papers p53 status determines the role of autophagy in Kriegsheim A, Humphries MJ, Norman JC, progression are activated in invasive human Laine A, Sihto H, Come C, Rosenfeldt MT, pancreatic tumour development. Nature 2013; 504: Caswell PT. squamous cell carcinoma. Am J Pathol 2013; 183: Zwolinska A, Niemela M, Khanna A, Chan EK, 296-300 RCP-driven alpha5beta1 recycling suppresses Rac 930-7 Kahari VM, Kellokumpu-Lehtinen PL, Sansom OJ, and promotes RhoA activity via the RacGAP1- Evan GI, Junttila MR, Ryan KM, Marine JC, Joensuu IQGAP1 complex. J Cell Biol 2013; 202: 917-35 Klejnot M, Gabrielsen M, Cameron J, Mleczak A, H, Westermarck J. Nick Morrice (page 51) Talapatra SK, Kozielski F, Pannifer A, Olson MF. Senescence sensitivity of breast cancer cells is Proteomics and Mass Spectrometry Muller PA, Trinidad AG, Caswell PT, Norman JC, Analysis of the human cofilin 1 structure reveals defined by positive feedback loop between CIP2A Vousden KH. conformational changes required for actin binding. and E2F1. Cancer Discov 2013; 3: 182-97 Mutant p53 regulates Dicer through p63 dependent Acta Crystallogr D Biol Crystallogr 2013; 69: 1780-8 Primary Research Papers and independent mechanisms to promote an Long JS, Crighton D, O’Prey J, Mackay G, Erazo T, Moreno A, Ruiz-Babot G, Rodriguez-Asiain invasive phenotype. J Biol Chem 2014; 289: 122-32 Lourenco FC, Munro J, Brown J, Cordero J, Zheng L, Palmer TM, Gottlieb E, Ryan KM. A, Morrice NA, Espadamala J, Bayascas JR, Gomez Stefanatos R, Strathdee K, Orange C, Feller SM, Extracellular adenosine sensing-a metabolic cell N, Lizcano JM. Other Publications Sansom OJ, Vidal M, Murray GI, Olson MF. death priming mechanism downstream of p53. Canonical and kinase activity-independent Rainero E, Norman JC. Reduced LIMK2 expression in colorectal cancer Mol Cell 2013; 50: 394-406 mechanisms for extracellular signal-regulated Late endosomal and lysosomal trafficking during reflects its role in limiting stem cell proliferation. kinase 5 (ERK5) nuclear translocation require integrin-mediated cell migration and invasion: cell Gut 2014; 63: 480-93

82 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 83 Ramirez-Peinado S, Leon-Annicchiarico CL, Capper D, Pritchard C, Inman GJ, Longerich T, Evan GI, Junttila MR, Ryan KM, Marine JC, Joensuu Radulescu S, Ridgway RA, Cordero J, Athineos D, Galindo-Moreno J, Iurlaro R, Caro-Maldonado A, Sansom OJ, Benitah SA, Zender L, Gil J. H, Westermarck J. Salgueiro P, Poulsom R, Neumann J, Jung A, Patel Prehn JH, Ryan KM, Munoz-Pinedo C. A complex secretory program orchestrated by the Senescence sensitivity of breast cancer cells is S, Woodgett J, Barker N, Pritchard DM, Oien K, Glucose-starved Cells Do Not Engage in inflammasome controls paracrine senescence. defined by positive feedback loop between CIP2A Sansom OJ. Prosurvival Autophagy. J Biol Chem 2013; 288: Nat Cell Biol 2013; 15: 978-90 and E2F1. Cancer Discov 2013; 3: 182-97 Acute WNT signalling activation perturbs 30387-98 differentiation within the adult stomach and rapidly Bird TG, Lu WY, Boulter L, Gordon-Keylock S, Lourenco FC, Munro J, Brown J, Cordero J, leads to tumour formation. Oncogene 2013; 32: Rosenfeldt MT, Bell LA, Long JS, O’Prey J, Nixon C, Ridgway RA, Williams MJ, Taube J, Thomas JA, Stefanatos R, Strathdee K, Orange C, Feller SM, 2048-57 Roberts F, Dufes C, Ryan KM. Wojtacha D, Gambardella A, Sansom OJ, Iredale Sansom OJ, Vidal M, Murray GI, Olson MF. E2F1 drives chemotherapeutic drug resistance via JP, Forbes SJ. Reduced LIMK2 expression in colorectal cancer Rajan P, Sudbery IM, Villasevil ME, Mui E, Fleming J, ABCG2. Oncogene epublished 25 Nov 2013, doi: Bone marrow injection stimulates hepatic ductular reflects its role in limiting stem cell proliferation. Davis M, Ahmad I, Edwards J, Sansom OJ, Sims D, 10.1038/onc.2013.470 reactions in the absence of injury via macrophage- Gut 2014; 63: 480-93 Ponting CP, Heger A, McMenemin RM, Pedley ID, mediated TWEAK signaling. Proc Natl Acad Sci U S A Leung HY. Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, 2013; 110: 6542-7 Ma Y, Li A, Faller WJ, Libertini S, Fiorito F, Gillespie Next-generation Sequencing of Advanced Prostate Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, DA, Sansom OJ, Yamashiro S, Machesky LM. Cancer Treated with Androgen-deprivation Ridgway R, Adams PD, Anderson KI, Gottlieb E, Cheung EC, Athineos D, Lee P, Ridgway RA, Lambie Fascin 1 is transiently expressed in mouse Therapy. Eur Urol published online 14 Aug 2013, doi: Sansom OJ, Ryan KM. W, Nixon C, Strathdee D, Blyth K, Sansom OJ, melanoblasts during development and promotes 10.1016/j.eururo.2013.08.011 p53 status determines the role of autophagy in Vousden KH. migration and proliferation. Development 2013; 140: pancreatic tumour development. Nature 2013; 504: TIGAR is required for efficient intestinal 2203-11 Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, 296-300 regeneration and tumorigenesis. Dev Cell 2013; 25: Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, 463-77 Mohseni M, Sun J, Lau A, Curtis S, Goldsmith J, Fox Ridgway R, Adams PD, Anderson KI, Gottlieb E, Other Publications VL, Wei C, Frazier M, Sansom O, Wong KK, Kim C, Sansom OJ, Ryan KM. Helgason GV, Holyoake TL, Ryan KM. Guest RV, Boulter L, Kendall TJ, Minnis-Lyons SE, Camargo FD. p53 status determines the role of autophagy in Role of autophagy in cancer prevention, Walker R, Wigmore SJ, Sansom OJ, Forbes SJ. A genetic screen identifies an LKB1/MARK signaling pancreatic tumour development. Nature 2013; 504: development and therapy. Essays Biochem 2013; Cell lineage tracing reveals a biliary origin of axis controlling the Hippo/YAP pathway. Nat Cell 296-300 55: 133-51 intrahepatic cholangiocarcinoma. Cancer Res Biol 2014; 16: 108-17 epublished 7 Dec 2013, doi: 10.1158/0008-5472. Tan EH, Morton JP, Timpson P, Tucci P, Melino G, Macintosh RL, Ryan KM. CAN-13-1911 Myant KB, Cammareri P, McGhee EJ, Ridgway RA, Flores ER, Sansom OJ, Vousden KH, Muller PA. Autophagy in tumour cell death. Semin Cancer Biol Huels DJ, Cordero JB, Schwitalla S, Kalna G, Ogg Functions of TAp63 and p53 in restraining the 2013; 23: 344-51 Karim SA, Creedon H, Patel H, Carragher NO, EL, Athineos D, Timpson P, Vidal M, Murray GI, development of metastatic cancer. Oncogene, Morton JP, Muller WJ, Evans TR, Gusterson B, Greten FR, Anderson KI, Sansom OJ. published online 22 Jul 2013, doi: 10.1038/ Sansom OJ, Brunton VG. ROS production and NF-kappaB activation onc.2013.287 Owen Sansom (page 36) Dasatinib inhibits mammary tumour development triggered by RAC1 facilitate WNT-driven intestinal Colorectal Cancer and Wnt Signalling in a genetically engineered mouse model. J Pathol stem cell proliferation and colorectal cancer Wiese KE, Walz S, von Eyss B, Wolf E, Athineos D, 2013; 230: 430-40 initiation. Cell Stem Cell 2013; 12: 761-73 Sansom O, Eilers M. The Role of MIZ-1 in MYC-Dependent Primary Research Papers Laine A, Sihto H, Come C, Rosenfeldt MT, Myant KB, Scopelliti A, Haque S, Vidal M, Sansom Tumorigenesis. Cold Spring Harb Perspect Med Acosta JC, Banito A, Wuestefeld T, Georgilis A, Zwolinska A, Niemela M, Khanna A, Chan EK, OJ, Cordero JB. 2013; 3: a014290 Janich P, Morton JP, Athineos D, Kang TW, Kahari VM, Kellokumpu-Lehtinen PL, Sansom OJ, Rac1 drives intestinal stem cell proliferation and Lasitschka F, Andrulis M, Pascual G, Morris KJ, Khan regeneration. Cell Cycle 2013; 12: 2973-7 Other Publications S, Jin H, Dharmalingam G, Snijders AP, Carroll T, Huels DJ, Cammareri P, Ridgway RA, Nobis M, Carragher NO, McGhee EJ, Morton JP, Medema JP, Sansom OJ. Sansom OJ, Anderson KI, Timpson P. Methods to assess Myc function in intestinal Advanced intravital subcellular imaging reveals vital homeostasis, regeneration, and tumorigenesis. three-dimensional signalling events driving cancer Methods Mol Biol 2013; 1012: 237-48 cell behaviour and drug responses in live tissue. FEBS J 2013; 280: 5177-97 Morton JP, Sansom OJ. MYC-y mice: from tumour initiation to therapeutic Nobis M, McGhee EJ, Morton JP, Schwarz JP, targeting of endogenous MYC. Mol Oncol 2013; 7: Karim SA, Quinn J, Edward M, Campbell AD, 248-58 McGarry LC, Evans TR, Brunton VG, Frame MC, Carragher NO, Wang Y, Sansom OJ, Timpson P, Phesse TJ, Sansom OJ. Anderson KI. Responding to R-spondin: slit2 potentiates intestinal Intravital FLIM-FRET imaging reveals dasatinib- regeneration. Cell Stem Cell 2013; 13: 512-4 induced spatial control of src in pancreatic cancer. Cancer Res 2013; 73: 4674-86 Steele CW, Jamieson NB, Evans TR, McKay CJ, Sansom OJ, Morton JP, Carter CR. Patel R, Gao M, Ahmad I, Fleming J, Singh LB, Rai Exploiting inflammation for therapeutic gain in TS, McKie AB, Seywright M, Barnetson RJ, Edwards pancreatic cancer. Br J Cancer 2013; 108: 997-1003 J, Sansom OJ, Leung HY. Sprouty2, PTEN, and PP2A interact to regulate prostate cancer progression. J Clin Invest 2013; 123: 1157-75

84 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 85 Douglas Strathdee (page 54) Dickens MP, Roxburgh P, Hock A, Mezna M, Kellam Sara Zanivan (page 40) Other Staff Transgenic Technology B, Vousden KH, Fischer PM. Vascular Proteomics 5-Deazaflavin derivatives as inhibitors of p53 Primary Research Papers ubiquitination by HDM2. Bioorg Med Chem 2013; Good JA, Wang F, Rath O, Kaan HY, Talapatra SK, Primary Research Papers 21: 6868-77 Primary Research Papers Cheung EC, Athineos D, Lee P, Ridgway RA, Lambie Neuhauser N, Nagaraj N, McHardy P, Zanivan S, Podgorski D, MacKay SP, Kozielski F. W, Nixon C, Strathdee D, Blyth K, Sansom OJ, Scheltema R, Cox J, Mann M. Optimized S-trityl-L-cysteine-based inhibitors of Hock AK, Lee P, Maddocks OD, Mason SM, Blyth K, Vousden KH. High performance computational analysis of kinesin spindle protein with potent in vivo antitumor Vousden KH. TIGAR is required for efficient intestinal large-scale proteome data sets to assess activity in lung cancer xenograft models. J Med iRFP is a sensitive marker for cell number and tumor regeneration and tumorigenesis. 2013; 25: incremental contribution to coverage of the human Chem 2013; 56: 1878-93 Dev Cell growth in high-throughput systems. Cell Cycle 463-77 genome. J Proteome Res 2013; 12: 2858-68 2013; 13: 220-6 Kaan HY, Major J, Tkocz K, Kozielski F, Other Publications Schiller HB, Hermann MR, Polleux J, Vignaud T, Rosenfeld SS. Maddocks OD, Berkers CR, Mason SM, Zheng L, Strathdee D, Whitelaw CB. Zanivan S, Friedel CC, Sun Z, Raducanu A, “Snapshots” of ispinesib-induced conformational Blyth K, Gottlieb E, Vousden KH. TT2013 meeting report: the Transgenic Gottschalk KE, Thery M, Mann M, Fassler R. changes in the mitotic kinesin Eg5. J Biol Chem Serine starvation induces stress and p53- Technology meeting visits Asia for the first time. beta1- and alphav-class integrins cooperate to 2013; 288: 18588-98 dependent metabolic remodelling in cancer cells. 2013; 22: 667-71 regulate myosin II during rigidity sensing of Transgenic Res Nature 2013; 493: 542-6 fibronectin-based microenvironments. Nat Cell Nagarajan S, Skoufias DA, Kozielski F, Pae AN. Biol 2013; 15: 625-36 Receptor-ligand interaction-based virtual screening Muller PA, Trinidad AG, Caswell PT, Norman JC, for novel Eg5/kinesin spindle protein inhibitors. J Marcos Vidal (page 38) Vousden KH. Zanivan S, Maione F, Hein MY, Hernandez-Fernaud Med Chem 2012; 55: 2561-73 Drosophila Approaches to Cancer Mutant p53 regulates Dicer through p63 dependent JR, Ostasiewicz P, Giraudo E, Mann M. and independent mechanisms to promote an SILAC-based proteomics of human primary Klejnot M, Falnikar A, Ulaganathan V, Cross RA, Primary Research Papers invasive phenotype. J Biol Chem 2014; 289: 122-32 endothelial cell morphogenesis unveils tumor Baas PW, Kozielski F. Lourenco FC, Munro J, Brown J, Cordero J, angiogenic markers. Mol Cell Proteomics 2013; 12: The crystal structure and biochemical Stefanatos R, Strathdee K, Orange C, Feller SM, Tan EH, Morton JP, Timpson P, Tucci P, Melino G, 3599-611 characterization of Kif15: a bifunctional molecular Sansom OJ, Vidal M, Murray GI, Olson MF. Flores ER, Sansom OJ, Vousden KH, Muller PA. motor involved in bipolar spindle formation and Reduced LIMK2 expression in colorectal cancer Functions of TAp63 and p53 in restraining the Zanivan S, Meves A, Behrendt K, Schoof EM, neuronal development. Acta Crystallographica reflects its role in limiting stem cell proliferation. development of metastatic cancer. Oncogene, Gut Neilson LJ, Cox J, Tang HR, Kalna G, van Ree JH, Section D, Biological Crystallography 2014; 70: 2014; 63: 480-93 published online 22 Jul 2013, doi: 10.1038/ van Deursen JM, Trempus CS, Machesky LM, 123-33 onc.2013.287 Linding R, Wickstrom SA, Fassler R, Mann M. Myant KB, Cammareri P, McGhee EJ, Ridgway RA, In vivo SILAC-based proteomics reveals Smith J, Larue L, Gillespie DA. Huels DJ, Cordero JB, Schwitalla S, Kalna G, Ogg Trinidad AG, Muller PA, Cuellar J, Klejnot M, Nobis phosphoproteome changes during mouse skin Chk1 is essential for the development of murine EL, Athineos D, Timpson P, Vidal M, Murray GI, M, Valpuesta JM, Vousden KH. carcinogenesis. Cell Rep 2013; 3: 552-66 epidermal melanocytes. Pigment Cell Melanoma Greten FR, Anderson KI, Sansom OJ. Interaction of p53 with the CCT complex promotes Res 2013; 26: 580-5 ROS production and NF-kappaB activation protein folding and wild-type p53 activity. Mol Cell triggered by RAC1 facilitate WNT-driven intestinal 2013; 50: 805-17 stem cell proliferation and colorectal cancer initiation. Cell Stem Cell 2013; 12: 761-73 Other Publications Berkers CR, Maddocks OD, Cheung EC, Mor I, Myant KB, Scopelliti A, Haque S, Vidal M, Sansom Vousden KH. OJ, Cordero JB. Metabolic Regulation by p53 Family Members. Cell Rac1 drives intestinal stem cell proliferation and Metab 2013; 18: 617-33 regeneration. Cell Cycle 2013; 12: 2973-7 Hock AK, Vousden KH. Stefanatos R, Bauer C, Vidal M. The role of ubiquitin modification in the regulation p120 catenin is required for the stress response in of p53. Biochim Biophys Acta 2014; 1843: 137-49 Drosophila. PLOS One 2013; 8: e83942 Kaelin W, Livingston D, Loda M, Vousden K, Mihich E. Twenty-fifth annual Pezcoller Symposium: Karen Vousden (page 20) Metabolism and tumorigenesis. Cancer Res 2013; Tumour Suppression 73: 6124-7

Primary Research Papers Muller PA. Cheung EC, Athineos D, Lee P, Ridgway RA, NCF2/p67phox: A novel player in the anti-apoptotic Lambie W, Nixon C, Strathdee D, Blyth K, Sansom functions of p53. Cell Cycle 2013; 12: 14 OJ, Vousden KH. TIGAR is required for efficient intestinal Muller PA, Vousden KH. regeneration and tumorigenesis. Dev Cell 2013; 25: p53 mutations in cancer. Nat Cell Biol 2013; 15: 2-8 463-77

86 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 87 Talapatra SK, Anthony NG, Mackay SP, Kozielski F. Sawicka M, Pawlikowski J, Wilson S, Ferdinando D, Theses Schachtner, Hannah (2013) Investigation of the Mitotic kinesin Eg5 overcomes inhibition to the Wu H, Adams PD, Gunn DA, Parish W. Cameron, Jenifer (2013) Investigating the role of functional and structural role of podosomes in phase I/II clinical candidate SB743921 by an The specificity and patterns of staining in human cofilin oxidation in cancer cell migration and megakaryocytes [PhD thesis, University of Glasgow, allosteric resistance mechanism. J Med Chem cells and tissues of p16INK4a antibodies invasion [PhD thesis, University of Glasgow, Beatson Institute] 2013; 56: 6317-29 demonstrate variant antigen binding. PLoS One Beatson Institute] 2013; 8: e53313 Smith, Joanne (2013) Role of checkpoint kinase 1 in Ulaganathan V, Talapatra SK, Rath O, Pannifer A, Derkits, Sahra (2013) Investigating the role of PTEN malignant melanoma [PhD thesis, University of Hackney DD, Kozielski F. Shah PP, Donahue G, Otte GL, Capell BC, Nelson and LKB1 in traditional and serrated mouse models Glasgow, Beatson Institute] Structural insights into a unique inhibitor binding DM, Cao K, Aggarwala V, Cruickshanks HA, Rai TS, of colorectal cancer [PhD thesis, University of pocket in kinesin spindle protein. J Am Chem Soc McBryan T, Gregory BD, Adams PD*, Berger SL*. Glasgow, Beatson Institute] Stefanatos, Rhoda (2013) Modelling tumourigenesis 2013; 135: 2263-72 Lamin B1 depletion in senescent cells triggers large- and the stress response in Drosophila melanogaster scale changes in gene expression and the Dou, Hao (2013) Mechanism of ubiquitin transfer by [PhD thesis, University of Glasgow, Beatson chromatin landscape. Genes Dev 2013; 27: 1787-99 RING E3 ligases [PhD thesis, University of Glasgow, Institute] Peter D. Adams (page 56) *corresponding authors Beatson Institute] Epigenetics of Cancer and Ageing Stevenson, Richard (2013) Investigating the role of Other publications Dowding, Sarah (2013) The regulation of RNA fascin in murine models of inflammatory bowel Lund K, Adams PD, Copland M. disease and intestinal tumourigenesis [PhD thesis, Primary Research Papers polymerase III-mediated transcription by p53, AP-1 EZH2 in normal and malignant hematopoiesis. University of Glasgow, Beatson Institute] Cruickshanks HA, McBryan T, Nelson DM, and JNKs [PhD thesis, University of Glasgow, 2014; 28: 44-9 Epub 2013/10/08 Vanderkraats ND, Shah PP, van Tuyn J, Singh Rai T, Leukemia Beatson Institute] Brock C, Donahue G, Dunican DS, Drotar ME, Pawlikowski JS, Adams PD, Nelson DM. Meehan RR, Edwards JR, Berger SL, Adams PD. Ferrari, Nicola (2013) Investigating Runx Senescence at a glance. 2013; 126: 4061-7 Senescent cells harbour features of the cancer J Cell Sci transcription factors in mammary gland epigenome. Nat Cell Biol 2013; 15: 1495-506 development and breast cancer [PhD thesis, University of Glasgow, Beatson Institute] Howard L, Mackenzie RM, Pchelintsev NA, Stephen Tait (page 60) McBryan T, McClure JD, McBride MW, Kane NM, Mitochondria and Cell Death Galbraith, Laura (2013) The role of cardiolipin in Adams PD, Milligan G, Baker AH. mitophagy [PhD thesis, University of Glasgow, Beatson Institute] Profiling of transcriptional and epigenetic changes Primary Research Papers during directed endothelial differentiation of Tait SW, Oberst A, Quarato G, Milasta S, Haller M, Klejnot, Marta (2013) Structural and Biochemical human embryonic stem cells identifies FOXA2 as a Wang R, Karvela M, Ichim G, Yatim N, Albert ML, insights into members of the kinesin and ubiquitin marker of early mesoderm commitment. Stem Cell Kidd G, Wakefield R, Frase S, Krautwald S, ligase families [PhD thesis, University of Glasgow, Res Ther 2013; 4: 36 Linkermann A, Green DR. Beatson Institute] Widespread Mitochondrial Depletion via Mitophagy Ivanov A, Pawlikowski J, Manoharan I, van Tuyn J, Does Not Compromise Necroptosis. Cell Rep 2013; Moreaux, Guenièvre (2013) Investigating Nelson DM, Rai TS, Shah PP, Hewitt G, Korolchuk 5: 878-85 VI, Passos JF, Wu H, Berger SL, Adams PD. downstream effectors of KRas signalling in vivo: Dusp6 and Fra1 [PhD thesis, University of Glasgow, Lysosome-mediated processing of chromatin in Other Publications Beatson Institute] senescence. J Cell Biol 2013; 202: 129-43 Tait, S. DNA: leukemia’s secret weapon of bone mass Macagno, Juan (2013) A study of focal adhesion Pawlikowski JS, McBryan T, van Tuyn J, Drotar destruction. Oncogene 2013; 32: 5199-200 ME, Hewitt RN, Maier AB, King A, Blyth K, Wu H, kinase in cancer using Drosophila melanogaster [PhD thesis, University of Glasgow, Beatson Adams PD. Tait SW, Green DR. Institute] Wnt signaling potentiates nevogenesis. Proc Natl Mitochondrial regulation of cell death. Cold Spring Acad Sci USA 2013; 110: 16009-14 Harb Perspect Biol 2013; 5: a008706 Muinonen-Martin, Andrew (2013) Melanoma cells Pchelintsev NA, McBryan T, Rai TS, van Tuyn J, induce LPA gradients that drive chemotactic Ray-Gallet D, Almouzni G, Adams PD. John Paul Career Award dispersal and invasion [PhD thesis, University of Placing the HIRA histone chaperone complex in the Glasgow, Beatson Institute] All third year PhD students at the Beatson are chromatin landscape. Cell Rep 2013; 3: 1012-9 eligible for this Award, named after Dr John Paul, Rosenfeldt, Mathias (2013) p53 and the role of the founding Director of the Institute. Candidates Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, autophagy in pancreatic cancer development [PhD prepare a progress report on their work and give a Mackay G, Mrowinska A, Au A, Rai TS, Zheng L, thesis, University of Glasgow, Beatson Institute] talk to staff and other students. Ridgway R, Adams PD, Anderson KI, Gottlieb E, Sansom OJ, Ryan KM. Roxburgh, Patricia (2013) Manipulating the p53 The winner of this year’s Award was Barbara p53 status determines the role of autophagy in pathway for cancer treatment [PhD thesis, Chaneton from Eyal Gottlieb’s group. Barbara’s pancreatic tumour development. Nature 2013; 504: University of Glasgow, Beatson Institute] work aims to characterise the metabolic 296-300 adaptations made by cancer cells with the goal of then targeting these as anti-cancer treatments.

88 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 89 CONFERENCES AND WORKSHOPS

Cancer Research UK Beatson researchers here as well as some of that planned for Open Evening For the second year in a row, we also offered 12 International Cancer Conference the recently opened Translational Cancer Research Our opening evening, which was held on 13th high school students the opportunity to visit us Targeting the Tumour Stroma Centre (TCRC) next door. Laura Machesky and March this year, attracted a large number of for a week in July to find out more about our work 7 - 10 July 2013 Kevin Ryan described ongoing projects in their attendees, particularly local school pupils. The and get some hands-on experience in the labs. Bute Hall, University of Glasgow groups, while Martin Drysdale gave an update on talks, given by Andrew Biankin (Director, TCRC), The feedback from this event was extremely Scientific Committee: Jim Norman, Jeff Evans, the progress of the Drug Discovery Programme, Owen Sansom (Deputy Director, Beatson positive and many thanks must go to Colin Nixon, Michael Olson, Owen Sansom, Marcos Vidal, which had its first quinquennial review this year. Institute), Jen Morton (Associate Scientist) and Emmanuel Dornier, Louise Mitchell, Amy Au, Sara Zanivan There were talks by Beatson Associates Daniel Max Nobis (PhD Student), focused on the topical Saadia Karim, Kirsteen Campbell, Emma Woodham, Murphy and Stephen Tait, who are relatively recent theme of personalised medicine and were Olivia Susanto, Catherine Barber and Ken Davies This year’s conference, which started a little earlier arrivals, and by Iain McNeish and David Chang from followed a series of lab tours and demos by our who all did a considerable amount of work to than previously, offered participants a packed and the TCRC who outlined their plans, which focus on hardworking volunteers. make this a success. exciting programme. On the first evening, the Colin ovarian and pancreatic cancer respectively. Thomson Memorial Keynote Lecture, sponsored by the Association for International Cancer Research, Poster for 2014 was given by Robert Kerbel (Toronto) who conference described his studies to develop better models for predicting the outcome of clinical trials. Throughout the four days, there was a range of other excellent speakers including Alexander Anderson, Neta Erez, Elisa Espinet, Yi Feng, Robert Insall, Jennifer Munson, Alexandra Naba, Juan Rodriguez-Manzaneque, Michael Samuel, Herbert Schiller and Viviana Vallacchi who gave selected short talks and Owen Sansom whose talk was sponsored by the journal Disease Models & Mechanisms. Neta Erez (Tel Aviv) was awarded the Portland Press sponsored prize for her talk describing work to characterise the role of cancer- associated fibroblasts in breast cancer progression. Following the poster session, Jean Albrengues (Nice), working on LIF cytokine and invasion, and Sir Paul Nurse FRS on a visit to Thomas Cox (Copenhagen), using proteome the Institute (image courtesy of Andreas Hock) analysis to study bone metastasis, were jointly awarded the AMSBIO-Trevigen sponsored prize. Visit by Royal Society President, Sir Paul The meeting was generously co-sponsored by Nurse FRS Cancer Research UK and the Association for Paul Nurse and members of the Royal Society International Cancer Research. visited the Institute on 1st November. Graduate students Emma Woodham, Linda Julian and The 2014 meeting will focus on the oncogenic Barbara Chaneton along with clinical research signals that initiate and regulate metabolic rewiring fellow Colin Steele gave talks about their work, as well as the adaptability of the metabolic network while graduate student Rhoda Stefanatos chaired a to stress. New therapeutic opportunities will also lively question and answer session with Paul and be discussed (see www.beatson.gla.ac.uk/conf around a hundred students and postdocs. Paul was for details). very generous with his time not only listening to the talks and quizzing the speakers but also then Beatson Retreat answering questions on a wide range of topics. It An Institute retreat was held in our lecture theatre was a thought provoking and inspiring event for th on 27 September, giving everyone a chance to all who attend, especially the junior researchers. hear about the latest, exciting work being done by

90 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE CONFERENCES AND WORKSHOPS 91 SEMINARS AT THE BEATSON INSTITUTE

The following seminars were held at the CR-UK Beatson June October Institute during 2013. Pascal Meier, The Breakthrough Toby Robins Joao Pedro de Magalhaes, Institute of Integrative Breast Cancer Research Centre, The Institute of Biology, University of Liverpool Cancer Research, London Jose Lizcano, Autonomous University of Jean–Philippe Theurillat, Dana-Farber Cancer January Andrew South, Division of Cancer Research, Barcelona, Spain Institute, Boston, USA University of Dundee Wolfgang Wick, Department of Neuro-oncology, Jason Locasale, Division of Nutritional Sciences, Christian Pilarsky, Medical Faculty “Carl Gustav University of Heidelberg, Germany Nigel Waterhouse, Queensland Institute for Cornell University, New York, USA Carus”, Technical University Dresden, Germany Medical Research, Brisbane, Australia Elton Zeqiraj, Samuel Lunenfeld Research Institute, David Bryant, Department of Anatomy University Tatjana Crnogorac-Jurcevic, Centre for Molecular Toronto, Canada Fabien Llambi, St. Jude Children’s Research of California, San Francisco, USA Oncology, Queen Mary University of London Hospital, Memphis, USA Paul Clarke, Division of Cancer Research, Jon Lane, School of Biochemistry, University of Julia Newton-Bishop, Section of Epidemiology University of Dundee Bristol and Biostatistics, University of Leeds Markus Ralser, Department of Biochemistry, July Bin-bing Zhou, Shanghai Jiaotong University University of Cambridge School of Medicine, China Elke Markert, Steve Kaiser, St Jude Children’s Research Hospital, April Oliver Hofmann, Bioinformatics Core, Harvard Department of Medicine and Center for Systems Memphis, USA School of Public Health, Boston, USA Biology, Rutgers Cancer Institute of New Sakari Vanharanta, Sloan-Kettering Institute, Chiara Gorrini, The Campbell Family Institute for Jersey, USA Ruth Muschel, CR-UK/MRC Gray Institute for New York, USA Radiation Oncology & Biology, University of Oxford Breast Cancer Research, Toronto, Canada John Stingl, Cancer Research UK, University of Neil Rodrigues, Boston University, USA Christine Watson, Department of Pathology, Cambridge Ghassan Mouneimne, Harvard Medical School, University of Cambridge Richard Youle, National Institutes of Health, February Boston, USA Bethesda, USA Rob Cairns, Princess Margaret Hospital, Melda Tozluoglu, CR-UK London Research Toronto, Canada Institute August November Ronen Zaidel-Bar, Mechanobiology Institute and Jurre Kamphorst, Lewis-Sigler Institute for Richard Gibbs, Human Genome Sequencing NUS Division of Bioengineering, Singapore Integrative Genomics, Princeton University, USA Center, Baylor College of Medicine, Houston, USA Pierre Leopold, Institute of Biology Valrose, Université Nice Sophia Antipolis, France Shehab Ismail, Max Planck Institute of Molecular Martin McMahon, Helen Diller Family Scott Lyons, CR-UK Cambridge Institute Comprehensive Cancer Center, University of Physiology, Dortmund, Germany Timour Baslan, Cold Spring Harbor Laboratory, Robert Goldman, Department of Cell and California, San Francisco, USA New York, USA Molecular Biology, Northwestern University, Elisabetta Dejana, Department of Biomolecular Chicago, USA Sciences and Biotechnologies, University of David Adams, The Wellcome Trust Sanger Milan, Italy Institute, London Helen Mott, University of Cambridge Krishnaraj Rajalingam, Institute of Biochemistry II, Marion MacFarlane, MRC Toxicology Unit, September Jane Endicott, Northern Institute for Cancer Leicester Goethe University School of Medicine, Frankfurt, Alexei Vazquez, Department of Radiation Research, Newcastle University Germany Kairbaan Hodivala-Dilke, Barts and The London Oncology and Center for Systems Biology, Juri Rappsilber, Wellcome Trust Centre for Cell School of Medicine and Dentistry Robert Wood Johnson Medical School, New Brunswick, USA Biology, University of Edinburgh David Tuveson, CSHL Cancer Centre, Cold Spring December Harbor Laboratory, New York, USA Clemens Schmitt, Max-Delbrück-Center for Patrizia Agostinis, KU Leuven, Belgium Molecular Medicine, Berlin, Germany Xin Lu, Ludwig Institute, Oxford March Thomas Cox, Biotech Research and Innovation Hannah Brown, Department of Oncology, May Centre, University of Copenhagen, Denmark University of Sheffield Martin Sos, Cellular & Molecular Pharmacology, Shankar Balasubramanian, Department of David Sabatini, Whitehead Institute, University of California, San Francisco School of Chemistry, University of Cambridge Cambridge, USA Medicine, USA

92 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE SEMINARS AT THE BEATSON INSTITUTE 93 STUDENTSHIPS AND POSTDOCTORAL ADMINISTRATION FELLOWSHIPS

The training and career development of students and staff is essential in As an independent charity it is important to ensure Scientific Administration that the Beatson Institute complies with all relevant Jackie Beesley, Catherine Winchester our mission to support cancer research of the highest standard. Our regulations and adopts best practice in financial, aim is to continue to attract enthusiastic scientists and clinicians early in personnel and corporate governance matters. Our The Scientific Administration team supports Finance & Human Resources team has been set up scientists at the Beatson Institute by taking minutes their careers to work with our established staff and to draw on their to do this, providing the Institute’s Board of at a range of scientific and administrative meetings, experience but also to spark new ideas in a stimulating research Governors with necessary financial and legal editing publications such as the Scientific Report, information, ensuring that funding bodies’ maintaining an up-to-date website, administering environment. As well as learning a very wide range of practical and administrative requirements are met and that the graduate student training programme, and technical skills our junior researchers participate in all intellectual funding for the Institute’s research activities is providing reports and answering queries for both properly managed. internal and external audiences, including Cancer activities and present and discuss their own work at internal seminars Research UK. The team also assists researchers in and external meetings. We provide support and facilities of the highest The Beatson Institute also needs to coordinate with identifying and applying for external grant funding the University of Glasgow’s central administration and has a role in providing oversight , training and standards and scientific interactions are encouraged by our over such matters as the administration of grants, advice in all aspects of research integrity. international conference, workshops and seminars and by funding payment of suppliers and staffing. Our Finance & Human Resources and Secretarial teams provide participation in external meetings. that vital link. Cancer Research Technology Maria Lopalco PhD Studentships administrative matters required in relation to the Finance & Human Resources Cancer Research Technology (CRT) is an The purpose of our clinician/graduate training University or funding body. The advisor gives Peter Winckles, Caroline Preacher oncology-focused technology transfer and programme is to give students and clinical fellows additional guidance by providing independent Jacqui Clare, Karen Connor, development company wholly owned by Cancer starting in research an opportunity to work in advice on any matters concerning the studentship Nicki Kolliatsas, Elaine Marshall, Frank Research UK with 130 employees based primarily state-of-the-art laboratories. This enables them to and by attempting to resolve any problems that McGeoghegan, Gary Niven, Lynn Wilson, in London and Cambridge. Since a substantial assess and develop their research talents to the full, may arise. Patricia Wylie amount of the funding for the Beatson Institute to decide whether a research career suits them and comes from Cancer Research UK, CRT manages to use their period of graduate study as a Postdoctoral Research Scientists and Fellows The Finance & Human Resources team is all intellectual property-related matters on behalf springboard for their future career path. Our We see postdocs as pillars of the research and responsible for all accounting and personnel of the Institute and the charity. To facilitate this, four-year studentships are designed to give intellectual activities of their own groups and of the management issues including banking, payments, there is a CRT Business Manager based full-time at graduates who show a strong aptitude for research Beatson Institute as a whole. The training grants management, budgeting, pay the Institute. the opportunity to complete substantial research programme is designed to promote the administration, staff policies and procedures, and projects resulting in very good publications. All development of outstanding and dedicated junior reporting financial information for funders, students receive training in safe working practices, scientists and we hope that by the end of their managers and the Board of Directors. writing project reports and other transferable skills. tenure they will be ready to compete for an Training also involves learning to be an independent independent position. Postdocs are initially scientist and students participate fully in the employed for three years but outstanding Secretarial intellectual life of the Institute, attending and giving individuals who are developing into independent Laraine Kernahan (PA to Professor Vousden), seminars and actively contributing to scientific scientists may be given additional support and Rebecca Gebbie, Barbara Laing, Catriona discussions. Students are also given the opportunity responsibility – such as more technical help or Lambert, Sarah Price to present posters on their work at national and mentoring a postgraduate student. At the discretion international conferences to enhance their network of their group leader, funding may be extended for The Secretarial team provides an extensive range of of scientific contacts. two more years. secretarial and office services. These include assisting with staff recruitment, organising travel Our students are registered at the University of For further details on Studentships, Postdoctoral and accommodation, seminar arrangements, Glasgow and are allocated a supervisor and an and other posts currently available, see our website organisation of our conferences and workshops, advisor who are jointly responsible for supporting www.beatson.gla.ac.uk. database maintenance and the running of the main and monitoring their performance and progress. reception for the Institute. The team plays an The supervisor is responsible for developing the www.glasgow.gov.uk and www.seeglasgow.com important role in maintaining internal links, and in student’s abilities, providing all practical support give general information about Glasgow and other relationships with Cancer Research UK, the required for the project and dealing with any useful links. University of Glasgow and many other organisations with which our scientists have contact.

94 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE ADMINISTRATION 95 THANKS FOR SUPPORTING US

The work of our various research groups would barely proceed without Marcos Vidal · Beattie NC3Rs (with OS) · Blair & Bryden Solicitors the substantial grant funding provided by Cancer Research UK to the · Margaret G Brown Beatson Institute and the University of Glasgow, now amounting to Karen Vousden · T Brown Association for International Cancer Research, · Cambridge Bioscience Ltd over £18 million per annum combined. We are also indebted to a European Community, Netherlands Organisation · David N Campbell number of other organisations that provide funding to our scientists, for Scientific Research, NHS Greater Glasgow & · Elizabeth M Campbell, in memory of Mr Clyde Health Board Endowment Fund, Rubicon, Alasdair Campbell usually supporting projects in a particular sphere of special interest, or West of Scotland Women’s Bowling Association · Cathcart Trinity Church, Art & Craft Club supporting the careers of talented junior scientists, enabling them to · Central District Grand Chapter, Order of Sara Zanivan the Eastern Star pursue their research interests within our laboratories. These Breast Cancer Campaign · Clyde Travel organisations, whose funding we appreciate greatly, are listed below. · Crookston Castle Primary School Beatson Associates · Holly Docherty, her friend Owen and The additional funding provided by these organisations makes possible her sister Carly much work that we otherwise could not be undertaking and has Peter Adams · Don Whitley Scientific Ltd BBSRC, Medical Research Council, NIH with · Dowling become integral and indispensable to our operations. National Institute on Aging & National Cancer · Dumbarton & District Probus Club Institute (USA) · Edrington · Noel Elliott Cancer Research UK Beatson Institute Laura Machesky · Enserve Technical Services LLP Association for International Cancer Daniel Murphy British Lung Foundation, European Community, · Fermentas (part of Thermo Fisher Scientific) Kurt Anderson Research, Breast Cancer Campaign, Medical · Glenfield Bowling Club Carestream, Technology Strategy Board Research Council Merck Sharp & Dohme · May Gow · J Graham Karen Blyth Nick Morrice Stephen Tait · Avril Haddow Royal Society Association for International Cancer Research ARC, BBSRC, EMBO, EU Marie Curie Actions, Royal Society · In memory of the late Alma Harper · Hendry, in memory of John and Jean Grant Martin Drysdale Jim Norman · RA Hockley, in memory of Dr Stuart Breast Cancer Campaign, CSIC (Spanish National We do not purposefully solicit contributions to our Medical Research Council (with MO) Gerrard Hoggar Research Council) work directly from the general public – we see this as the role of the cancer charities such as those that · Hodder & Stoughton Ltd Jeff Evans · Mary Hopkin, in memory of her husband Bill Scottish Executive – Chief Scientist Office, Michael Olson feature above. We are, however, fortunate to be in · E Irving, in memory of her husband Medical Research Council, Pancreatic Cancer Breast Cancer Campaign, Medical Research the minds of many local people and organisations · The James Inglis Trust Research Fund, University of Glasgow Council (with MD) who give generously of their time and effort to raise funds for good causes. We are also, more · Barbara Jordan · Kelvinside Hillhead Parish Church, in memory Kevin Ryan poignantly, in the minds of those who are suffering Eyal Gottlieb of Dr Stuart Gerrard Hogger AIRC (Italian Association for Cancer Research), Association for International Cancer Research, cancer, or who have lost loved ones to this disease. · Emily Sharpe, Demlza Hart, Khyra Cannon FEBS, IAP programme (Belgian Science Policy), EMBO To those who give time and effort to raise funds on our behalf and to those who thoughtfully regard and Parmis Taghizadeh of Killermont Janssen Pharmaceutica NV, Metabomed Primary, Bearsden Owen Sansom us as suitable beneficiaries of their generosity, thank you. · Lanarkshire Women’s Bowling Association Robert Insall Association for International Cancer Research, · Rita Lindsay Wellcome Trust Bioven, EMBO, European Community, Institute of · Legacy of Mr Magnus John Mackay Cancer Research, Medical Research Council, · Aggreko UK Ltd AMV Ltd · Legacy of Miss Grace F MacLean Hing Leung Medimmune, NC3Rs (with MV), Royal Society, · · E McCrum The Academy of Medical Sciences, Medical Wellcome Trust · Anachem Ltd · H McCulloch Research Council, Prostate Cancer Charity, · Legacy of the late Mary Paterson Dunning · Christina McDougall, in memory of her Emma Shanks Anwyl-Jones University of Glasgow husband John Breast Cancer Campaign · Staff of Aviva Insurance W McGuineness, in memory of Mrs Isabel Adam · Bearsden Primary School ·

96 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE THANKS FOR SUPPORTING US 97 PATRONS AND BOARD OF GOVERNORS

Patrons

His Grace the Duke of Hamilton The Rt Hon. Lord Mackay of Clashfern The Viscountess Weir

Board of Directors

The Beatson Institute is an autonomous charity, constituted as a company limited by guarantee, registered in Scotland. The Institute is governed by its Board of Directors who are the directors of the company and trustees of the charity. The Board is ultimately responsible for all aspects of the Institute, including its scientific strategy, operational policies, regulatory compliance and financial stewardship · Ian McHale and members of 1 Section at MOD · Members, friends and family of St Rollox and accountability. On a day-to-day basis, many of Police, RNAD Coulport Bowling Club these responsibilities are delegated to the Institute’s · Fiona and Sarah McNeill, M&S Simply Food · In memory of Mr Bob Sharp Management Team. Store, Bearsden · In memory of the late Aline Legros · Fiona McNeill and family · In memory of the late Duncan MacLullich Prof Nic Jones (Chair) · B Millar · StarLab (UK) Ltd Chief Scientist, Cancer Research UK · Mosshead Primary School · Edward Stephen, in memory of his wife Mrs · Mrs Agnes Hill, in memory of Aline Legros Mary Johanna Stephen Mr Craig Anderson · Murray, Gillies & Wilson · Stratech Scientific Ltd Senior Partner, KPMG · Employees of Nationwide Building Society · Strathblane Bowling Club · Christine Neale · Children of Struther Hill Activity Group Mr Ian Dickson · North View Housing Association · Mr & Mrs Sweeney Consultant, MacRoberts Solicitors, Glasgow · Novus Bio · Synantix · Anne O’Hare, Hillpark Bowling Club and Hillpark · John Teevan, in memory of his late mother Dr Iain Foulkes Ladies Bowling Section · The Local Charity Shop Executive Director, Strategy and Research Funding, · Dr & Mrs J D Olav Kerr’s Charitable Trust · The West Coast Motors Cancer Research UK · Worthy Matron, Worthy Patron, Office Bearers · Thornhill Gardening Society and Members of the Order of the Eastern Star, Lily · UK Labs Direct Ltd Mr Ian Kenyon of the Valley · J Walker Chief Financial Officer, Cancer Research UK · Order of the Eastern Star, Priory Chapter No 21 · H Wardell, in memory of her husband Mr · Pacitti Jones Anthony Richard Wardell Company Secretary · Maurice Paterson · Mr & Mrs Webb, in memory of Mr Philip Mr Peter Winckles · PEQLAB Ltd Alfred James Cancer Research UK Beatson Institute · Sarah Percy and Irene Kennedy · West of Scotland Women’s Bowling · Promega UK Ltd Association D & M Wiseman · Gordon Ronney, in memory of Ian O’Friel from Dr Wilma Gemmell Trust · Rotary Club of Clydebank · Rutherglen Vogue Celtic Supporters Club · GCH Shepherd · P Sommerville · Source Bioscience UK Ltd · St Andrew of Glasgow Royal Arch Chapter No.69

98 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE PATRONS AND BOARD OF GOVERNORS 99 CONTACT DETAILS

The Beatson Institute for Cancer Research ISSN 1740-732X Director: Professor Karen H Vousden Copyright © 2012 Cancer Research UK Tel: +44(0) 141 330 3953 and University of Glasgow Fax: +44(0) 141 942 6521

Address Cancer Research UK Beatson Institute Garscube Estate, Switchback Road Bearsden, Glasgow G61 1BD

e-mail: [email protected] web site: www.beatson.gla.ac.uk

Registered address The Beatson Institute for Cancer Research Inland Revenue Charity Ref. SC006106 Registered as a company limited by guarantee in Scotland No. 84170 Registered address: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD

Tel +44(0) 141 330 3953 www.beatson.gla.ac.uk

Electronic version of this report can be found at: www.beatson.gla.ac.uk/annual_report

Cancer Research UK Cancer Research UK is a registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103). Registered address: Angel Building, 407 St John Street, London EC1V 4AD

Tel 44(0) 20 1234 5678 www.cruk.org

100 SCIENTIFIC REPORT 2013 CANCER RESEARCH UK BEATSON INSTITUTE Cancer Research UK Beatson Institute Garscube Estate Switchback Road Bearsden Glasgow G61 1BD Telephone +44(0) 141 330 3953 www.beatson.gla.ac.uk

www.beatson.gla.ac.uk