SCIENTIFIC REPORT 2014 cruk.org SCIENTIFIC Cover image PhD student Christin Bauer from Marcos Vidal’s lab, who uses the fruit fly as a tool to study cancer biology. REPORT 2014 BEATSON INSTITUTE CONTENTS

SECTION 1 DRUG DISCOVERY INTRODUCTION 04 Martin Drysdale 48 RESEARCH HIGHLIGHTS 06 Drug Discovery Programme BACKGROUND 10 ADVANCED TECHNOLOGIES CANCER RESEARCH UK BEATSON INSTITUTE Kurt Anderson 52 Beatson Advanced Imaging Resource (BAIR) REGULATION OF CANCER CELL GROWTH Gabriela Kalna 53 METABOLISM AND SURVIVAL Bioinformatics and Computational Biology Eyal Gottlieb 12 Gillian Mackay 54 Tumour Metabolism Metabolomics Danny Huang 14 Gaurav Malviya 55 Ubiquitin Signalling Nuclear Imaging Hing Leung 16 Sergio Lilla and David Sumpton 56 Prostate Cancer Biology Proteomics and Mass Spectrometry Kevin Ryan 18 Emma Shanks 57 Tumour Cell Death RNAi Screening Alexei Vazquez 20 Karen Blyth 58 Mathematical Models of Metabolism Transgenic Models of Cancer Karen Vousden 22 Douglas Strathdee 59 Tumour Suppression Transgenic Technology

REGULATION OF CANCER CELL INVASION SECTION 2 AND METASTASIS BEATSON ASSOCIATES Kurt Anderson 26 Peter D. Adams 62 Tumour Cell Migration Epigenetics of Cancer and Ageing Jeff Evans 28 David Bryant 64 Translational Cancer Therapeutics Molecular Control of Epithelial Polarity Robert Insall 30 Jurre Kamphorst 66 Cell Migration and Chemotaxis Cancer Metabolomics Shehab Ismail 32 Daniel J. Murphy 68 Structural Biology of Cilia Oncogene-Induced Vulnerabilities Cancer Research UK Beatson Laura Machesky 34 Institute building Stephen Tait 70 Migration, Invasion and Metastasis Mitochondria and Cell Death Jim Norman 36 Integrin Cell Biology SECTION 3 Michael Olson 38 RESEARCH FACILITIES 74 Molecular Cell Biology PUBLICATIONS 78 Owen Sansom 40 CONFERENCES AND WORKSHOPS 90 Colorectal Cancer and Wnt Signalling SEMINARS 92 Marcos Vidal 42 STUDENTSHIPS AND POSTDOCTORAL 94 Drosophila Approaches to Cancer FELLOWSHIPS Sara Zanivan 44 ADMINISTRATION 95 Vascular Proteomics THANKS FOR SUPPORTING US 96 PATRONS AND BOARD OF GOVERNORS 99 CONTACT DETAILS 100

2 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE 3 INTRODUCTION

The success of the Beatson Institute continued this year, with Professor Robert Insall and The activities supported by the Centre range Professor Laura Machesky who from curating core resources to developing became Fellows of the Royal outstanding quinquennial reviews for four of our largest methods, providing advice and expertise, and Society of Edinburgh in March groups and continued recruitment into both core funded and building capacity to undertake specific projects. These activities encompass a wide variety of University of Glasgow supported positions. expertise and different operational structures, necessitating the development of individualised models for resource management and access. A number of key appointments this year mean Two of our senior group leaders, Robert Insall This is an ongoing process, with a major focus Professor Karen Vousden that the Institute is now at almost full capacity. and Laura Machesky were recognised for their to date on preclinical trials and functional CBE, FRS, FRSE, FMedSci We welcomed new group leaders, Shehab considerable contributions to their fields by screens. A number of calls for bids to take Director of the Cancer Ismail and Alexei Vazquez along with our final being elected as Fellows of the Royal Society of Research UK Beatson advantage of these new means of support have Institute two Beatson Associates, David Bryant and Jurre Edinburgh, while Jeff Evans was made a Fellow led to the approval of several exciting new Kamphorst. Heather McKinnon was also of the Royal College of Physicians of Edinburgh. projects aimed at translating our basic science. appointed as Head of Biology in Drug Discovery. I was also honoured to give the Norman Heatley Lecture at the University of Oxford and the Finally, huge thanks must to go our many Shehab is establishing a programme focused on Inaugural Women of Distinction in Science and supporters who both remember us in the form the structural biology of cilia, while Dave is Medicine Lecture at Queen Mary University of of legacies and work so hard to raise funds for studying the control cell polarity in cancer. London, while junior group leader Jurre us, including especially Clyde Travel, Mosshead Meanwhile, with their respective expertise in Kamphorst was awarded a prestigious Cancer Primary School and the West of Scotland lipid metabolism and computational modelling, Research Career Development Award. Postdoc Women’s Bowling Association who do this so Jurre and Alexei considerably strengthen our Robert Fordham was awarded a Royal tirelessly and to such effect year after year. Cancer Metabolism Research Unit (CaMeRU). Commission for the Exhibition of 1851 The Unit also received vital funding from the Fellowship, while postdoc Kevin Myant moved Beatson Endowment for equipment. across to Edinburgh to establish an independent research group. We said farewell to Nick Morrice, head of our proteomics facility and for the time being, To further develop our expertise in cancer Sergio Lilla and David Sumpton are jointly metabolism, we were delighted to welcome running this important service for us. My PA experts on cancer metabolism to our Laraine Kernahan also retired from the Institute conference In July. The conference, led by Eyal in August and Sheila McNeill has joined us to Gottlieb and Jim Norman, was a great success take on this key role. and allowed us to introduce new and old friends to Glasgow and the Beatson. Four of our senior scientific groups -Eyal Gottlieb, Jim Norman, Mike Olson and Karen Recruitment to the Cancer Research UK Vousden - had highly successful reviews this Glasgow Centre’s infrastructure posts is now year, while we were also delighted with Karen also largely complete and includes the Blyth’s well deserved promotion to Senior Staff appointment of Liz Musgrove, the new Synergy Scientist. As head of our transgenic facility, Manager for the Centre, who has overall Karen is a vital component of our mouse responsibility for coordinating the infrastructure models team and she was enthusiastically and developing mechanisms through which all supported by our external expert review panel. Centre members can access it and Fiona Thomson, senior research fellow in cancer All of the Beatson faculty are now working hard biomarkers/pharmacology and Director of the to attract grant funding from various sources to Analytical Services Unit. support our work. In several cases this has also involved establishing new collaborations and networks that further broaden our expertise and research areas.

4 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE INTRODUCTION 5 RESEARCH HIGHLIGHTS

This section features some of the key research findings made by scientists at the Beatson Institute and Institute of Cancer Sciences in the past year.

Beatson Institute Labuschagne CF, van den Broek NJ, Mackay GM, Vousden KH, Maddocks OD. Cordero JB, Ridgway RA, Valeri N, Nixon C, Serine, but not glycine, supports one-carbon Frame MC, Muller WJ, Vidal M, Sansom OJ. metabolism and proliferation of cancer cells. c-Src drives intestinal regeneration and Cell Rep 2014; 7: 1248-58 transformation. EMBO J 2014; 33: 1474-91 Building on previous work showing that cancer Src is overexpressed or activated in up to 20% of cells can be highly dependent on serine/glycine colorectal tumours, although it is unclear what uptake for proliferation, this study uses a new the consequences of this might be. To begin to technique called Pulse-Stop-Flux, which address this, the authors investigate Src actions detects flux into low-abundance metabolites, to within the intestine of the Drosophila and the determine whether cancer cells can use serine mouse. They show that Src is necessary and and glycine interchangeably. The authors sufficient to drive intestinal stem cell conclude that serine, rather than glycine proliferation during tissue self-renewal, consumption supports both nucleotide regeneration and tumourigenesis, and that this synthesis and cancer cell proliferation. occurs via upregulation of EGFR and activation Morran DC, Wu J, Jamieson NB, Mrowinska A, Muinonen-Martin AJ, Susanto O, Zhang Q, of Ras/MAPK and Stat3 signalling. Kalna G, Karim SA, Au AY, Scarlett CJ, Chang Smethurst E, Faller WJ, Veltman DM, Kalna G, Li A, Morton JP, Ma Y, Karim SA, Zhou Y, Faller DK, Pajak MZ, Oien KA, McKay CJ, Carter CR, Lindsay C, Bennett DC, Sansom OJ, Herd R, WJ, Woodham EF, Morris HT, Stevenson RP, Gillen G, Champion S, Pimlott SL, Anderson KI, Jones R, Machesky LM, Wakelam MJ, Knecht Faller WJ, Jackson TJ, Knight JR, Ridgway RA, Juin A, Jamieson NB, MacKay CJ, Carter CR, Evans TR, Grimmond SM, Biankin AV, Sansom DA, Insall RH. Jamieson T, Karim SA, Jones C, Radulescu S, Leung HY, Yamashiro S, Blyth K, Sansom OJ, OJ, Morton JP. Melanoma cells break down LPA to establish Huels DJ, Myant KB, Dudek KM, Casey HA, Machesky LM. Targeting mTOR dependency in pancreatic local gradients that drive chemotactic dispersal. Scopelliti A, Cordero JB, Vidal M, Pende M, Fascin is regulated by slug, promotes cancer. Gut 2014; 63: 1481-9 PLoS Biol 2014; 12: e1001966 Ryazanov AG, Sonenberg N, Meyuhas O, Hall progression of pancreatic cancer in mice, and is MN, Bushell M, Willis AE, Sansom OJ. associated with patient outcomes. In this study, the authors use established Metastasis is particularly prevalent in melanoma, mTORC1-mediated translational elongation Gastroenterology 2014; 146: 1386-96 e1-17 preclinical models of pancreatic cancer to leading to high mortality rates. This work seeks limits intestinal tumour initiation and growth. demonstrate that tumours driven by activated to understand what drives melanoma cells to Nature 2015; 517: 497-500. doi: 10.1038/ This paper provides strong evidence that the KRAS and PTEN loss are dependent on mTOR migrate out of a tumour with such efficiency nature13896. Epub 2014 Nov 5 actin-bundling protein fascin is regulated by signalling and are uniquely responsive to its and what role, if any chemotaxis plays in this. the transcription factor slug and involved in inhibition, when compared to tumours driven Using a chamber-based assay developed in the In a series of in vivo experiments, the authors of pancreatic tumour formation and metastatic by activated KRAS and mutant p53. They also lab, the authors show that melanoma cell this paper demonstrate that mTORC1 is an spread. Mechanistically, the authors show show that approximately 20% of human dispersal occurs by positive chemotaxis as a essential downstream effector of Wnt signalling that fascin promotes filopodia formation and pancreatic tumours have low PTEN expression result of outward-facing lysophosphatidic in the intestine. They show that it is absolutely cell invasion. They also find an association and a gene expression signature similar to that acid (LPA) gradients. Importantly, cells in vivo required for enterocyte proliferation following between fascin levels and patient outcomes seen in the preclinical model, leading them to and in culture generate these gradients APC loss and Wnt activation, and that this is as a suggesting fascin could be a useful marker or conclude that anti-mTOR therapies may benefit themselves by breaking down LPA. This result of it increasing translation elongation. therapeutic target. this subset of patients. Overall, this work leads the authors to conclude that melanoma Thus, the authors suggest that targeting mTOR illustrates how, in the future, genetic signatures drives its own metastasis. and translation control might benefit patients at of human tumours could be used to personalise high risk of developing colorectal cancer. cancer treatments, making them more effective.

6 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH HIGHLIGHTS 7 RESEARCH HIGHLIGHTS (CONTINUED)

Rosenfeldt MT, Bell LA, Long JS, O’Prey J, Nixon mutant p53, p63 and p73. As well as confirming C, Roberts F, Dufes C, Ryan KM. that mutant p53 binds to both p63 and p73, the E2F1 drives chemotherapeutic drug resistance authors show that MDM2 preferentially binds to via ABCG2. Oncogene 2014; 33: 4164-72 p73 rather than p63. This leads them to propose a model in which MDM2 competes with p63 for In this paper, the authors report that, in a binding to mutant p53 to restore p63 activity, number of settings, E2F1 regulates the while forming a trimeric complex with p73 and expression of the transporter ABCG2 leading to mutant p53 to more strongly inhibit p73 drug efflux and suppression of chemotherapy- function. The study highlights the complex induced cell death. They also find that elevated interactions that can occur between p53 family E2F1 correlates with ABCG2 expression in members and the proteins that regulate them. human lung cancer. Thus, the study highlights an exciting new function for this transcription factor, which is frequently upregulated in van den Biggelaar M, Hernandez-Fernaud JR, ATG5. The authors find that ATG12 is highly MYC, an oncogene frequently overexpressed in cancer, in multidrug resistance and cancer van den Eshof BL, Neilson LJ, Meijer AB, unstable and targeted for proteasomal cancer, can drive both cell proliferation and therapy. Mertens K, Zanivan S. degradation via ubiquitination. As a apoptosis. The latter limits its oncogenic Quantitative phosphoproteomics unveils consequence of this turnover, ATG12 potential and the authors of this paper set out to temporal dynamics of thrombin signaling in contributes to proteasome inhibitor-mediated determine, using a single transgenic model, Scopelliti A, Cordero JB, Diao F, Strathdee K, human endothelial cells. Blood 2014; 123: apoptosis. Since proteasome inhibitors are used what the key mediator of this MYC-induced White BH, Sansom OJ, Vidal M. e22-36 as anti-cancer agents, this may be a clinically apoptosis is. They conclude that the BH3-only Local control of intestinal stem cell important finding. protein BIM, rather than p19ARF, is required for homeostasis by enteroendocrine cells in the As well as being the key enzyme involved in MYC-induced apoptosis. Thus, BH3 mimetics adult Drosophila midgut. Curr Biol 2014; 24: coagulation, thrombin triggers PAR1 signalling might prove useful therapeutically in boosting 1199-211 in the endothelium. In this paper, the authors Lund K, Cole J, VanderKraats ND, McBryan T, MYC’s intrinsic apoptotic response. combine SILAC, phosphopeptide enrichment Pchelintsev NA, Clark W, Copland M, Edwards Enteroendocrine cells in the intestine are known techniques and mass spectrometry to analyse JR, Adams PD. to translate local cues into systemic responses thrombin-induced signalling in human primary DNMT inhibitors reverse a specific signature of Rai TS, Cole JJ, Nelson DM, Dikovskaya D, Faller by releasing hormones into the bloodstream. endothelial cells. They identify 2224 thrombin- aberrant promoter DNA methylation and W, Vizioli MG, Hewitt RN, Anannya O, McBryan However, using the Drosophila midgut as a regulated phosphorylation sites, providing a associated gene silencing in Acute Myeloid T, Manoharan I, van Tuyn J, Morrice N, model system, the authors of this study identify unique resource for future studies of thrombin Leukemia. Genome Biol 2014; 15: 406 Pchelintsev NA, Ivanov A, Brock C, Drotar M, a novel, local role for these cells as paracrine and PAR signalling. Ultimately, a greater Nixon C, Clark W, Sansom OJ, Anderson KI, regulators of intestinal stem cell proliferation. understanding of this signalling could lead to In this paper, the authors use whole genome King A, Blyth K, Adams PD. Furthermore, they show that this is mediated via the development of improved PAR1 antagonists, DNA methylation and RNA sequencing to HIRA orchestrates a dynamic chromatin Bursicon/DLGR2 signalling, leading to stem cell for use as anti-coagulants, that affect platelet identify a set of genes whose methylation and landscape in senescence and is required for quiescence. The authors suggest these results but not endothelial cell function. silencing in AML is reversed by DNA suppression of neoplasia. Genes Dev 2014; 28: point toward a potential role for LGRs as tumour methyltransferase inhibitors. The authors 2712-25 suppressor genes. conclude that, as good candidates for direct Institute of Cancer Sciences regulation by DNA methyltransferase inhibitors, Cellular senescence acts as an important barrier reactivation of these genes might contribute to to proliferation and tumour progression, and Stindt MH, Muller PA, Ludwig RL, Kehrloesser S, Haller M, Hock AK, Giampazolias E, Oberst A, therapeutic activity as well as making them extensive changes to chromatin occur in Dotsch V, Vousden KH. Green DR, Debnath J, Ryan KM, Vousden KH, useful biomarkers. senescent cells. However, the molecular basis Functional interplay between MDM2, p63/p73 Tait SWG. and consequences of these changes is not fully and mutant p53. Oncogene 2014 Nov 24. doi: Ubiquitination and proteasomal degradation of understood. The authors of this study show that 10.1038/onc.2014.359. [Epub ahead of print] ATG12 regulates its pro-apoptotic activity, Muthalagu N, Junttila MR, Wiese KE, Wolf E, non-proliferating senescent cells express and Autophagy 2014; 10: 2269-78 Morton J, Bauer B, Evan GI, Eilers M, incorporate H3.3 and other histones into a MDM2 binds to and is an important regulator of Murphy DJ. dynamic chromatin landscape. Importantly, wild type p53, the tumour suppressor protein. This study investigates the regulation of the BIM is the primary mediator of MYC-induced they also find that the histone chaperone HIRA However, this study examines the functional essential autophagy and ubiquitin-like protein apoptosis in multiple solid tissues. Cell Rep 2014; is required for regulation of this landscape and consequences of MDM2’s interactions with ATG12 when in its free state, not conjugated to 8: 1347-53 for suppression of oncogene-induced neoplasia in vivo.

8 SCIENTIFIC REPORT 2014 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 Cancer Research UK Beatson Institute BEATSON INSTITUTE

Eyal Gottlieb - Tumour Metabolism Danny Huang - Ubiquitin Signalling Hing Leung - Prostate Cancer Biology Kevin Ryan - Tumour Cell Death Alexei Vazquez - Mathematical Models of Metabolism Karen Vousden - Tumour Suppression

10 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE REGULATION OF CANCER CELL GROWTH METABOLISM AND SURVIVAL 11 in the kidney demonstrated that FH deletion in TUMOUR METABOLISM the kidney causes benign cysts but fails to www.beatson.gla.ac.uk/eyal_gottlieb induce renal cell carcinoma (RCC) (Zheng et al., Cancer Metab 2013; 1: 12). Using primary non-immortalised kidney cells from these mice, we recognised that acute FH deletion leads to redox stress-induced senescence and p21 induction (Fig. 2). We further demonstrated that fumarate accumulation in FH-deficient cells leads to oxidative stress by covalently binding and inactivating reduced glutathione (GSH) forming a succinic-GSH adduct (Fig. 2). This Our lab utilises state-of-the-art metabolomics capabilities to metabolite was found in both human and mouse FH-deficient cells and in kidney cysts of study metabolic transformations and identify metabolic Fh1-deficient mice. We also demonstrated that vulnerabilities in cancer. An early hallmark of cancer tissues is the oxidative stress induced by GSH succination is sufficient to elicit cellular senescence in metabolic reprogramming, first noted by Otto Warburg who non-transformed cells. Importantly, the ablation found that cancer cells rely on glycolysis under aerobic Figure 1 of cancer cells under metabolically stressful of p21, a key mediator of senescence, in Fh1- Acetate metabolism under conditions (Schug et al., Cancer Cell 2015; deficient mice resulted in the transformation of conditions. More recent research showed that metabolic metabolic stress. The depletion 27: 57). benign renal cysts into a hyperplastic lesion, of available oxygen and fatty suggesting that fumarate-induced senescence alterations in cancer involve many additional pathways, acids to cancer cells growing at a Group Leader needs to be bypassed for the initiation of renal potentially increasing the number of clinical targets. In fact, distance from the blood supply Glutathione succination by fumarate induces Eyal Gottlieb induces a hypoxia adaptive redox stress and cellular senescence and is a cancers (Zheng et al., Nature Commun 2015; 6: most, if not all tumour suppressors and oncogenes regulate response and de novo fatty acid molecular barrier that protect FH-deficient 6001). These results suggest that fumarate- biosynthesis, two metabolic Research Scientists renal cells from carcinoma induced senescence may be a novel tumour hallmarks observed in most Simone Cardaci1 metabolism. Furthermore, tumours are typically placed in a suppressive event that must be circumvented human cancers. However, Mutations in the tricarboxylic acid (TCA) cycle Nadja Pfetzer2 metabolically stressful environment, leading to essential metabolic adaptations to these enzyme, fumarate hydratase (FH) are associated for RCC formation. It is worth noting that Zachary Schug3 stressful conditions require a HLRCC patients also develop multiple benign Saverio Tardito with hereditary leiomyomatosis and renal cell metabolic adaptations. Our major interest is in metabolic switch of nutrient utilisation, Liang (Leon) Zheng cancer (HLRCC) syndrome, which is kidney cysts that then evolve into aggressive turning from glucose to acetate characterised by the formation of mostly benign malignant carcinoma. Furthermore, in many enzymes that also function as tumour suppressors or as a major carbon source for Scientific Officer human RCCs, other than HLRCC, low FH fatty acid biosynthesis. This smooth muscle tumours and, in some patients, Elaine MacKenzie oncogenes or those which regulate the essential metabolic metabolic switch is mediated by a highly malignant form of papillary and expression is associated with disease ACSS2. progression. Hence, recognising the Clinical Research Fellow requirements of cancer cells. collecting duct renal cell cancer. Although the Stefan Nowicki4 mechanisms of tumourigenesis related to FH mechanisms that lead FH-deficient cells to mutations have been extensively investigated, overcome senescence in vivo will help us Graduate Students Acetyl-CoA synthetase 2 promotes acetate targets, ACSS2 exhibited the highest level of little is known about the overall consequences understand and hopefully treat human RCC Elodie Kuntz utilisation and maintains cancer cell growth amplification in invasive breast carcinomas (7%). that loss of FH has on cellular metabolism. A in general. Laura Galbraith under metabolic stress The characterisation of acetate metabolism Jiska van der Reest mouse model of FH conditional gene deletion The metabolic adaptation of cancer cells to during hypoxia by heavy isotope tracing Publications listed on page 80 1 FEBS environmental changes often involves an coupled with liquid chromatography-mass

2 increase in the uptake and metabolism of spectrometry (LC-MS) and nuclear magnetic Janssen Pharmaceutica Figure 2 extracellular resources such as glucose, amino resonance (NMR) technologies confirmed that 3 CRUK Discovery Fumarate induces glutathione Committee/Astra Zeneca acids and lipids. However, cancer cells can acetate was consumed by cancer cells in an succination and senescence. (A) 4 CRUK Glasgow Centre further modify or shift their metabolism during ACSS2-dependent manner. It also showed that The loss of FH leads to fumarate periods of nutrient stress. Over the last two acetate was used to synthesise fatty acids and accumulation, which forms years, we led a consortium of five groups, TCA cycle intermediates. In contrast, the adducts with the thiol group of GSH (succinic-GSH). (B) Redox co-funded by a CRUK Discovery Committee formation of these metabolites from glucose- stress mediated by glutathione award and AstraZeneca, with the specific derived acetyl-CoA was significantly diminished succination leads to p21 purpose of identifying targets in the field of lipid under hypoxia. These results suggested that induction and benign renal cysts metabolism. We performed siRNA screens on a when mitochondrial glucose oxidation was in an Fh1-deficient mouse focused set of 60 genes using a panel of 13 compromised (hypoxia) or when the availability model. (C) The co-ablation of the p21 gene in the Fh1-deficient breast and prostate cancer cell lines. Cells were of lipids was limited (low serum), acetate was mouse model leads to hyper- maintained in either 10% or 1% serum, and in needed to generate acetyl-CoA for the proliferation as indicated by both normoxic and hypoxic conditions. Under production of energy (TCA cycle) and biomass increase Ki67 staining of conditions of 1% serum and hypoxia, the (fatty acids/cholesterols). These mechanistic hyperplastic regions. expression of acetyl-CoA synthetase 2 (ACSS2), effects were reinforced by phenotypic studies an enzyme that converts acetate into showing that ACSS2 silencing inhibited the acetyl-CoA in many of the cancer cell lines, was growth in two-dimensional cultures, thre- markedly induced. Accordingly, silencing of dimensional spheroids and tumour xenografts ACSS2 strongly inhibited cellular growth under (Fig. 1). In summary, our data revealed a these conditions. Data mining and disease previously unappreciated role for acetate as a linkage analyses revealed that of our shortlisted nutritional source for the growth and survival

12 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR METABOLISM 13 the proteasome or via endocytosis. Although our results showed that LHR Tyr371 UBIQUITIN SIGNALLING Independent of their E3 activity, Cbls also phosphorylation could induce dramatic www.beatson.gla.ac.uk/danny_huang function as adaptor proteins through conformational changes to activate c-Cbl, interactions with a variety of proteins involved in these changes are insufficient to account for diverse biological processes. In RTK signalling the 1400-fold enhancement in catalytic cascades, Cbls act as both positive and negative efficiency. More recently we showed that regulators: they propagate signals downstream phosphorylation of LHR Tyr plays an additional of activated RTKs as adaptors and crucial role. By determining a crystal structure simultaneously ubiquitinate and promote of phosphoTyr363-Cbl-b (Tyr363 is the degradation of the same RTKs as E3s. Recent corresponding LHR Tyr in Cbl-b) bound to a studies showed that c-Cbl mutations are found TKBD substrate peptide and an E2 covalently in human patients with myeloproliferative linked to Ub (E2~Ub), we showed that the Post-translational modification of ubiquitin (Ub) initiated by diseases and these mutations abrogate E3 ligase phosphoTyr-induced E2-binding surface also activity and induce cell transformation participates in Ub binding (Fig. 2b). Notably, the sequential actions of Ub-activating enzyme (E1), Ub- (reviewed in Kale et al., Cancer Res 2010; 70: phosphate moiety of the phosphoTyr363 conjugating enzyme (E2) and Ub ligase (E3) regulates diverse 4789). It remains elusive how Cbls are regulated directly interacts with Ub’s Thr9 sidechain (Fig. and how these mutations could contribute 2c). These Ub interactions alone enhance Cbl’s cellular processes, including signal transduction, cell cycle to oncogenicity. catalytic efficiency by ~200-fold, explaining progression, apoptosis and gene transcription. Deregulation in the massive rate enhancement upon Figure 2 All Cbls share a highly conserved N-terminal phosphorylation (Dou et al., Nat Struct Mol the Ub pathway is often associated with human pathogenesis, Phosphorylated LHR tyrosine SH2-containing tyrosine kinase-binding domain Biol 2013; 20: 982). participates activation of E2~Ub. (TKBD), a linker helix region (LHR) and a RING including cancer. (a) The conserved Cbl’s Group Leader domain (Fig. 2a) followed by a variable proline- c-Cbl’s Ty371 is one of the mutational hotspots N-terminal domain containing Danny Huang TKBD, LHR and RING domain. rich region (PRR). The TKBD mediates substrate in patient with myeloid neoplasms. Our results Our group uses X-ray crystallography and of human cancer cells, and are currently in (b) Structure Tyr363 specificity by binding to proteins containing explain why Tyr371 mutants cannot adopt active Research Scientists biochemical approaches to study the enzymes clinical trials. In addition, several E3 inhibitors phosphorylated Cbl-b bound to phosphotyrosine motifs commonly found in Cbl configuration and optimise E2~Ub for Lori Buetow in the Ub pathway to understand their have been developed. Our group is interested in an E2~Ub and a TKBD substrate RTKs or tyrosine kinases whereas the PRR transfer. We are currently investigating on peptide. (c) Close-up view of Mads Gabrielsen regulation, mechanistic functions and understanding the regulation and mechanistic recruits proteins containing an SH3 domain. The how c-Cbl mutations could alter c-Cbl’s pTyr363-Ub and RING-Ub mutation-induced deregulation. We anticipate functions of RING E3s with particular focus on LHR and RING domain play central roles in conformation transitions and contribute Scientific Officer interactions. All structures are that the knowledge gained from our structural RING E3s that have been linked to cancer. Gary Sibbet coloured as in (a). E2 is in light recruiting E2s and in mediating target to oncogenicity. studies will assist in the development of blue, TKBD substrate peptide is in ubiquitination. To gain insights into the Graduate Students selective therapeutic targets within the Cbl proteins and receptor tyrosine pink and Ub is in green. regulation of Cbls, we determined three new Mechanism of Ub transfer by RING E3 Dominika Kowalczyk Ub pathway. kinase signalling crystal structures of c-Cbl: native c-Cbl, c-Cbl RING E3s recruit E2 thioesterified with Ub Gabriele Marcianò Activation of receptor tyrosine kinase (RTK) bound to a TKBD substrate peptide and c-Cbl (E2~Ub) and substrate to facilitate the Ub Amrita Patel Ubiquitin conjugation cascade signalling cascades is important for cellular phosphorylated at Tyr371 in complex with an E2 transfer from E2 to the amino group of a Covalent attachment of Ub involves three key processes such as cell proliferation, and a TKBD substrate peptide. Our structures substrate lysine. How RING E3s promote Ub enzymes, namely E1, E2 and E3 (Fig. 1). E1 differentiation, migration and survival. and the existing structure of c-Cbl bound to an transfer is unclear. We have now determined initiates the cascade by adenylating Ub’s Prolonged or aberrant activation of RTKs is E2 and a TKBD substrate peptide (Zheng et al., two crystal structures of RING E3 bound to E2 C-terminus in the presence of Mg2+ and ATP, commonly associated with cancer. Cbl proteins Cell 2000; 102: 533) reveal dramatic UbcH5B covalently linked to Ub at its active site. followed by the formation of a covalent (Cbls), c-Cbl, Cbl-b and Cbl-c are RING E3s that conformational changes in the LHR and RING Both structures reveal extensive noncovalent thioester intermediate with Ub. E1 then recruits negatively regulate RTKs, tyrosine kinases and a domain. We showed that in the donor Ub interactions with UbcH5B and the an E2 and transfers the thioesterified Ub to the host of other proteins by promoting their unphosphorylated state, c-Cbl adopts an RING domain. Notably an additional Ub-binding E2’s catalytic cysteine. E3 plays a pivotal role in ubiquitination and subsequent degradation by auto-inhibited conformation where its element outside the RING domain is crucial in determining substrate fate. In general, E3 E2-binding surface on the RING domain is stabilising the Ub’s Ile36 surface (Fig. 3). In consists of an E2-binding module (HECT, RING occluded in a competitive manner to reduce E2 dimeric RING E3s such as BIRC7 and RNF4, the Figure 1 or U-box domain) and a protein-protein binding, thereby attenuating c-Cbl’s activity. We C-terminal tail of the second RING subunit acts Enzymatic cascade for Ub interaction domain that confers substrate found that Tyr371 phosphorylation enhances as this additional Ub-binding component, and modifications. specificity. With this configuration, E3 recruits E2 c-Cbl’s catalytic efficiency by 1400-fold. Tyr371 in Cbl-b, phosphorylation of LHR Tyr363 creates thioesterified with Ub and substrate to promote phosphorylation activates its ligase activity by a phosphoTyr-induced element adjacent to the Ub transfer from the E2 to a lysine side chain on inducing dramatic LHR conformational changes RING domain that functionally mimics that the substrate. In humans, the Ub pathway that (1) enhance overall E2 binding affinity by dimeric RING E3 tail in stabilising Ub. Both consists of two E1s, ~30-40 E2s and ~600 E3s eliminating auto-inhibition and forming a new Ub-binding components optimise kcat and Km that collectively ubiquitinate thousands of phosphoTyr371-induced E2-binding surface; for Ub transfer. These results demonstrate that, different substrates. The Ub pathway has and (2) place the RING domain and E2 in in addition to Ub-E2, Ub-RING and E2-RING emerged as the target for therapeutic proximity of the substrate-binding site. We interactions, an additional Ub-binding intervention. Velcade, a proteasome inhibitor, is showed that phosphoTyr371-induced component outside the RING domain is the best example and is currently used for Figure 3 conformational transition is required for EGFR essential for stabilisation of the donor Ub Requirements for E2~Ub treating patients with multiple myeloma and activation for transfer by RING ubiquitination. Together these results configuration, restraining the globular Ub into a relapsed mantle cell lymphoma. Studies E3s. An additional component demonstrate how Tyr371 phosphorylation could closed conformation and allowing optimal demonstrated that inhibitors of NEDD8 E1 (highlighted in yellow) outside transiently switch on Cbl’s ligase activity to positioning of Ub’s C-terminus for transfer (Dou (Soucy et al., Nature 2009; 458: 732) and E2 the RING domain (orange) is attenuate RTK signalling (Dou et al., Nat Struct et al., Nat Struct Mol Biol 2012; 19: 876; Dou et Cdc34 (Ceccarelli et al., Cell 2011; 145: 1075) required for stabilisation of Ub by Mol Biol 2012; 19: 184). al., Nat Struct Mol Biol 2013; 20: 982). Cbl (a) and by the dimeric RING induced apoptosis and suppressed proliferation E3s (b).

14 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE UBIQUITIN SIGNALLING 15 We undertook quantitative tumour PROSTATE CANCER BIOLOGY transcriptome profiling pre- and post-ADT by www.beatson.gla.ac.uk/hing_leung mRNA-Seq on prostate tumours from eight patients with locally advanced/metastatic prostate cancer (Rajan et al., Eur Urol 2014; 66: 32). We identified 774 upregulated and 755 downregulated ADT responsive genes, with enrichment for upregulated genes within the Wnt/β-catenin-signalling pathway. We observed enhanced β-catenin immunoreactivity in a subset of castration-resistant prostate cancer. Using the isogenic LNCaP/LNCaP-AI human Prostate cancer is a major global health issue and a Figure 1 Figure 2 prostate cancer cell lines as models for very common cause of morbidity and mortality in men hormone naïve and castration-resistant disease Figure 1 prostate cancer. Applying the androgen receptor respectively, we demonstrated expression worldwide. Our research programme builds on ongoing Overview of the interdependent positive human CWR22 prostate cancer cell line, changes consistent with mRNA-Seq data nature of our research we genetically suppressed SPRY2 expression by among selected members of the pathway, work on aberrant cellular signalling (including androgen methodology and approach. stably transfecting an shRNA construct to target including the Frizzled family of receptors (FZD4/ receptor, Sprouty2 and ERK5) and novel preclinical models Figure 2 SPRY2 mRNA. Using the CWR22 SPRY2 KD cells FZD5/FZD7/FZD8) and the downstream effector to fast track the development of new treatment agents or Summary of three PTEN- as orthografts in a nude mouse model, their JUN. Upon treatment with the tankyrase interacting events to promote growth characteristics were determined inhibitor XAV939, to promote β-catenin prostate carcinogenesis. strategies for prostate cancer through cross-disciplinary following surgical castration, with control animals degradation, LNCaP-AI cell growth was more Group Leader collaborations (Fig. 1). subjected to mock surgery. Orthografts of potently suppressed when compared to the Hing Leung CWR22 SPRY2 KD cells showed minimal parental LNCaP cells. shrinkage despite castration, contrasting to the We further genetically engineered the Clinical Lecturers Here, we select three examples (Fig. 2) where interest with upregulated mRNA and protein dramatic response shown by the control, non- Imran Ahmad Pb-Cre:Ptenfl/+ CatnbΔ(ex3)/+ mice, which form Null silencing shRNA (NSi). Hence, CWR22 1 we strive to integrate our laboratory and clinical expression levels in the prostates of SB:Pten Prabhakar Rajan aggressive tumours by 6-8 months and have a resources to provide new biologic insight into mice, which also showed upregulation of the SPRY2 KD represents castration-resistant prostate cancer. significantly poorer survival outcome compared Associate Scientist castration-resistant prostate cancer for future downstream Pparg target genes such as Fasn, to control mice (Pb-Cre:CatnbΔ(ex3)/+). The Rachana Patel clinical applications. Acc, Acly (key lipogenic enzymes) at protein and Pb-Cre:Ptenfl/flCatnb Δ(ex3)/+ mice with mRNA levels. Similarly, in human PC3 cells, While surgical castration clearly suppressed Research Scientist serum testosterone levels as expected, we found homozygous loss of Pten have an even shorter 2 In vivo sleeping beauty (SB) transposon screen PPARG-targeting siRNA suppressed the Yan Zhou that the intra-tumoural testosterone levels in latency, suggesting Pten has a ‘dose dependent’ identified Pparg activation as a key interacting expression of these PPARG targets. Oil Red O SPRY2 KD orthografts were maintained. effect in driving tumour progression. Scientific Officer event with Pten lost in driving prostate staining demonstrated a reduction in levels of Furthermore, the expression of key enzymes for Importantly, Pb-Cre:CatnbΔ(ex3)/+ mice develop Janis Fleming tumourigenesis through altered triglycerides and cholesterol esters in the siRNA androgen biosynthesis were significantly elevated prostate cancer by 12 months indicating that lipid metabolism knockdown cell line. Reduced PPARG Technicians/Research stabilisation of β-catenin is sufficient to drive [Collaborators: Owen Sansom, Dave Adams expression in PC3 cells significantly suppressed in the CWR22 SPRY2 KD orthografts, signifying de Associates novo androgen synthesis upon castration. We tumourigenesis, while heterozygous loss of Carolyn Loveridge (Wellcome Trust Sanger Centre, Cambridge) proliferation and migration at 24 hours (p<0.001 then subjected orthografts of CWR22 SPRY2 KD Pten (Pb-Cre:Ptenfl/+) does not result in Jacqueline Stockley3 Sioban Fraser and Joanne Edwards and p<0.005, respectively). prostate cancer. (Histopathology, University of Glasgow)] cells (along with non-silencing (Nsi) control cells) Clinical Research Fellows In a clinical cohort of prostate cancer, nuclear to castration (as a means to achieve androgen Lisa Bashford deprivation therapy, ADT) or treatment with The molecular basis for upregulated β-catenin 4 Mutations in the tumour suppressor PTEN has PPARG expression was upregulated in prostate Mark Salji simvastatin, alone or in combination, in a expression in clinical prostate cancer remains to 4 been associated with the development and cancer (all Gleason grades) compared to the Sarah Slater be fully investigated. We found that the RNA progression of clinical prostate cancer. benign controls (p<0.001). Importantly, the four-week treatment regime. Combined ADT and binding protein, hnRNPA2 may regulate Graduate Student Transgenic mice carrying homozygous deletion presence of upregulated PPARG expression and simvastatin resulted in a significantly stronger β-catenin protein expression and is itself Elspeth Brzezinska in Pten develop prostate cancer after a long low levels of PTEN was associated with a shorter tumour killing response in CWR22 SPRY2 KD cells, despite their ability to survive ADT alone. overexpressed in prostate cancer (Stockley et al., 1 latency (>6 months) suggesting that additional disease specific survival when compared to RCSEng / CRUK The level of nuclear androgen receptor was RNA Biol 2014; 11: 755). Both nuclear and Clinician Scientist genetic ‘events’ are required (Ahmad et al., Proc those patients with only low PTEN (median: 7.05 effectively abolished by combined treatment, cytoplasmic hnRNPA2 protein are present and 2 until September Nat Acad Sci 2011; 108: 16392). Male mice with versus 2.05 years; p=0.0015), consistent with along with significant levels of apoptosis appear to be functionally important in prostate 3 RCSEng / CRUK appropriate SB genotype: SB:PtenNull (Pb- data from our in vivo screen. (enhanced cleaved caspase 3 expression). Even in cancer. Cytoplasmic hnRNPA2 may upregulate 4 MRC Clinical Research Cre4:Ptenfl/flT2Onc3/+Uren/+) and littermate the control cells with NSi, which signified β-catenin protein expression and modulate Fellowship controls [PtenNull (Pb-Cre4:Ptenfl/fl) and SBControl Sprouty2 deficiency promotes castration- hormone responsive disease, the tumour cellular phenotype through 3’-UTR-mediated (Pb-Cre4:T2Onc3/+Uren/+)] were generated and resistant prostate cancer through de novo response was enhanced following combined effects. aged. SB:PtenNull mice exhibited significantly androgen synthesis ADT and statins treatment. Based on these accelerated prostate tumourigenesis with larger [Collaborator: Owen Sansom] exciting early data, we are initiating efforts to Summary tumours and enhanced metastasis. We have recently shown that loss of PTEN Taken together, we continue to make excellent cooperates with Sprouty2 (SPRY2) deficiency by design a clinical study to directly test the value of combining statins with currently available ADT. progress in our understanding of key events in Overall, 59 unique common insertion sites (CIS) bypassing key tumour suppressor checkpoints aggressive (and treatment-resistant) prostate were identified from theSB:Pten Null cohort, and (Patel et al., J Clin Invest 2013; 123: 1157). cancer. Through this new knowledge and our using a cross-species oncogenomics approach, Analysing a panel of matched hormone naïve The Wnt/β-catenin-signalling pathway drives novel model systems, we are developing and six CIS fulfilled a predetermined set of criteria for and castration-resistant resected prostate prostate carcinogenesis and may mediate designing new therapeutic strategies. further validation. Pparg (isoform 1), a critical tumours, SPRY2 expression was suppressed resistance to androgen deprivation therapy [Collaborators: Chris Ponting (University of regulator of lipid metabolism, is of particular significantly further in castration-resistant Oxford), Owen Sansom] Publications listed on page 81

16 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE PROSTATE CANCER BIOLOGY 17 Figure 1 A novel technique to measure TUMOUR CELL DEATH p73-mediated activation of A2B autophagic flux primes cells to die in response to In addition to apoptosis, autophagy is another www.beatson.gla.ac.uk/kevin_ryan accumulation of extracellular adenosine. Activation of p73 by a process that can regulate cell viability. As the chemotherapeutic drug leads to name suggests, autophagy is a process of increased expression of the gene self-eating whereby cytoplasmic constituents encoding the adenosine are engulfed in double membrane structures receptor, A2B. In the presence of called autophagosomes that serve to deliver elevated levels of extracellular adenosine, expression of A2B cargoes to lysosomes for degradation. causes upregulation of Puma and cell death by apoptosis. Under basal conditions, autophagy acts to preserve cellular integrity by promoting the The aim of our group is to understand the factors Our further analysis revealed that the cell death degradation of damaged and misfolded response from p73 and adenosine was proteins and organelles. Over time, autophagy is regulating cell viability in cancer. Since it is known that apoptotic and involved upregulation of the therefore very important for both cellular and inhibition of cell death mechanisms is a common event in BH3-only protein, PUMA leading to organismal viability. Autophagy is, however, also mitochondrial outer membrane highly adaptable and a variety of internal and tumour development, this poses problems for many forms of permeabilisation (MOMP) (Fig. 1). external cues can alter the rate of autophagic chemotherapy that utilise cell death pathways, leading to degradation. For example, a phylogenetically In our studies relating to p53 and A2B, we conserved autophagic response occurs when drug resistance. We are investigating both known cell death observed that both cisplatin and hypoxia cause cells are deprived of nutrients. Under these regulators as well as searching for novel proteins and pathways accumulation of extracellular adenosine, which conditions, autophagic degradation is increased Group Leader that control cell viability and chemosensitivity. We envisage can be sensed by elevated levels of A2B to and degradation of cellular constituents is Kevin Ryan promote cell death. We were therefore keen to enhanced to boost ATP levels during the that the knowledge gained from our studies will be translated identify chemotherapeutic agents or conditions starvation period. Importantly, this response is Research Scientists that could activate ADORA2B expression and/or also thought to occur during the starvation Alice Baudot1 and lead to the improvement of existing clinical regimens or cause accumulation of adenosine that could be periods that occur during tumour evolution and Florian Beaumatin sensed by p73-induced A2B. To exclude the there is good evidence that certain tumours 2 new targets for therapeutic intervention. Damian Graczyk involvement of p53, these studies were depend on autophagy both as a way to respond Sanket Joshi3 Jaclyn Long1, 3 undertaken in HCT116 cells in which p53 had to metabolic stress and as a means to preserve Jun-ichi Sakamaki3 Metabolic cell death priming The tumour suppressor p53 is the most been deleted by homologous recombination. cellular fidelity. downstream of p73 frequently inactivated gene in human cancer. Figure 2 In these cells, we found that Adriamycin, but not Scientific Officer It is now widely accepted that tumour cells Approximately 50% of tumours have a p53 Enhanced mitophagy can be cisplatin causes a p73-dependent induction of In light of the key roles of autophagy, it is James O’Prey undergo changes in metabolism to facilitate mutation and it is considered that many other used to measure autophagy flux. ADORA2B expression. In addition, we found important to have reliable tools to study the Expression of exogenous Parkin Graduate Students growth and mitigate the oxidative stress tumours contain mutational events in other that exposure of these cells to ultraviolet light process. For example, since drugs that target causes autophagic degradation Michaela Mrschtik associated with anabolism. It therefore seemed genes that affect either p53 activation or causes a highly significant increase in autophagy are being developed, it is important of mitochondria en masse when Pablo Sierra Gonzalez conceivable that tumour suppressive downstream effectors of the p53 response. This cells are treated with agents that extracellular adenosine, which enhanced to know in any given scenario if autophagy is mechanisms should exist that detect these naturally caused us to consider whether cause mitochondrial damage. p73-induced death in a manner dependent on being enhanced, impaired or even blocked. To 1 Worldwide metabolic changes and invoke a cellular ADORA2B (the gene encoding A2B) was also This provides the cell with an A2B. These studies therefore not only broaden facilitate research in this area, we have Cancer Research response to either correct the change or activated by other p53 family proteins such that unlimited pool of autophagic the range of transcription factors that can developed an assay that we consider reliably 2 EMBO Fellowship eradicate the damaged cell. This idea led to our this metabolic cell death priming mechanism substrate such that the rate of induce metabolic cell death via A2B but they measures the autophagic flux or capacity of 3 Astellas Pharma Inc loss of mitochondrial proteins previous discovery that the tumour suppressor could be active in the absence of wild type p53. can be used to assess the also increase the spectrum of scenarios in cells. This assay involves ectopic expression of p53 activates the expression of the adenosine autophagy rate/capacity of which adenosine can be produced to signal the ubiquitin ligase, Parkin, which enables receptor, A2B (Long et al., Mol Cell 2013; 50: p73 is a protein closely related to p53 both in the cell. tumour cell death. autophagic degradation of mitochondria en 394). We found that enhanced expression of structure and function, and our studies revealed masse when cells are treated with agents that A2B alone has little impact on the cell but if that it too can activate expression of ADORA2B. cause mitochondrial damage (Fig. 2). When this expressed in the presence of high levels of Activation was observed with the three occurs, the simple assessment of the rate of loss extracellular adenosine (the receptor’s natural transactivating isoforms - TA-p73α, TA-p73β and of certain mitochondrial proteins can be used as ligand), then a potent cell death response TA-p73γ – but not with the dominant-negative a read-out of the underlying autophagic rate ensues. The importance of this cell death form of p73, ΔN-p73. Interesting, activation by within the cell. Using this assay, we have been priming mechanism to tumour suppression the TA-p73s occurred through the same DNA able to detect the differences in autophagic flux becomes clear when we consider that high binding element that is activated by p53, between different cells and the changes in levels of adenosine can be derived from the indicating that activation of ADORA2B is a autophagy that occur upon treatment with breakdown of the energy-rich molecule, ATP. conserved response (Fig. 1). autophagy modulators or upon genetic ablation Under conditions of metabolic stress, such as of an essential autophagy gene. As a result, we occurs in a growing tumour, the energy-rich Another indication of the conservation of this consider this assay to be an important new tool phosphate bonds in ATP are broken and response came from our analysis of cell death in for those investigating the role of autophagy in adenosine is produced. This adenosine the presence of adenosine. This revealed that tumour cell integrity and tumour cell death. accumulates outside the cell, and studies have expression of TA-p73β and TA-p73γ, but shown that the extracellular space of solid interestingly not TA-p73α, sensitised cells to the Publications listed on page 83 tumours can be bathed in adenosine. presence of extracellular adenosine and that this sensitisation was lost upon depletion of A2B.

18 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR CELL DEATH 19 MATHEMATICAL MODELS OF METABOLISM www.beatson.gla.ac.uk/alexei_vazquez

Figure 1 Metabolism is essential for life and its alteration is implicated in The construction of a genome multiple human diseases. The transformation from a normal scale mathematical model of metabolism requires the cell to a cancerous one requires metabolic changes to fuel the specification of three major ingredients: the nutrients that are high metabolic demands of cancer cells, including but not present in the extracellular media, the set of biochemical limited to cell proliferation and cell migration. Our group reactions that are encoded by the genome of the cells under investigates cancer metabolism from an evolutionary point of study and the cell metabolic view. We hypothesise that given specific microenvironmental objective. We use this core Group Leader model to investigate how conditions and metabolic constraints, there is an optimal metabolic constraints and Alexei Vazquez evolutionary principles shape mode of cell metabolism to achieve a metabolic objective. the differential utilisation of This metabolic mode will offer an evolutionary advantage and metabolic pathways. therefore be selected for during the time course of cancer development. First, we aim to uncover the metabolic objectives and metabolic constraints upon which natural selection is acting. Second, we aim to determine which known (or yet to be discovered) molecular alterations are driving the deterministic or stochastic occurrence of the optimal metabolic modes.

Impact of molecular crowding on cancer remodelling status of cancer cells determines cell metabolism their differential response to anticancer drugs Molecular crowding constrains the abundance that target metabolism. We plan to investigate of enzymes, ribosomes and organelles in the the interplay between proliferation and tissue cell cytoplasm. Our previous work indicates that remodelling during cancer evolution. this constraint limits the metabolic capabilities of cells and shapes the differential utilisation of Drugs targeting cancers with misfolded metabolic pathways. Cancer cells are subject to mutant p53 molecular overcrowding due to the We have identified a small molecule that overexpression of amplified genes and the specifically targets cancer cells carrying a accumulation of mutated protein aggregates. mutant form of the p53 gene, a master regulator We plan to investigate the impact of this of cellular responses to stress. This molecule molecular overcrowding on cancer metabolism acts specifically on a subset of mutant p53 and determine its relevance to anticancer proteins that are partially misfolded. The ability therapy. of these misfolded p53 proteins to recover their wild type function is modulated by the small Proliferation and tissue remodelling are two molecule, the zinc levels in cells and, potentially, major metabolic programmes in cancer by metabolic status. We plan to investigate the Our previous work indicates that proliferation interplay between the metabolic status of cells and tissue remodelling are major drivers of gene and their response to small molecules that expression patterns in cancer tissues, and they modulate misfolded p53 proteins. determine cancer subtypes with significant survival differences. Furthermore, analysis ofin Publications listed on page 86 vitro data indicates that the proliferation/

20 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE MATHEMATICAL MODELS OF METABOLISM 21 we have previously shown that this interaction TUMOUR SUPPRESSION does not result in the ubiquitylation and www.beatson.gla.ac.uk/karen_vousden degradation of p73. In agreement with previous publications, we found that the MDM2/p63 interaction was much weaker. Interestingly, we showed that MDM2 can inhibit p63 binding to p53R175H but enhanced the weaker p53R273H/ p73 interaction. These effects on the interactions are reflected in an ability of MDM2 to relieve the inhibition of p63 by p53 R175H, but enhance the inhibition of p73 activity by p53 R175H and R273H. We have therefore proposed Over the past year, we have extended the analysis of one a model in which MDM2 competes with p63 for binding to p53 R175H to restore p63 activity, but carbon metabolism in cancer cells and are beginning to forms a trimeric complex with p73 and p53 develop in vivo cancer models to test new therapeutic R273H to more strongly inhibit p73 function. These interactions are likely to be affected and combinations that include limitation of dietary serine for modified further by other binding partners, such effects on tumour cell growth. as HSP70, and the impact on the formation of Figure 1 serine in various mouse models. Our results these different complexes remains to be fully Under fully fed conditions (LHS), would also suggest that depriving cancers of explored. However, our results reveal an cells use serine to produce We have also made progress in understanding showed that higher concentrations of glycine serine would inhibit their growth, and over the interesting differential activity of MDM2 on the glycine and feed the one carbon Group Leader the role of MDM2 on the activity of tumour led to a further inhibition of cell proliferation - cycle, allowing for the synthesis past year we have been able to confirm that activity of mutant p53, suggesting that the ability Karen Vousden derived mutant p53s. Finally, our work on an observation that was not fully explained by a of purines and NADPH, and feeding mice a serine depleted diet resulted in of different mutant p53s to inhibit p63 and p73 is USP42, a deubiquitinating enzyme that we non-functional glycine cleavage system. To providing precursors for the SAM reduced tumour growth in both xenograft and modulated by the expression of MDM2. CBE, FRS, previously showed targets p53, has expanded to understand this response we developed a new cycle that is important for the genetically engineered cancer models. While methylation of DNA and protein. reveal an exciting activity of USP42 in controlling method for measuring metabolic flux, which we these results validate our general approach, they We have been very interested in the regulation FRSE, FMedSci In the absence of serine (RHS), gene expression more generally. termed ‘pulse-stop-flux’. Using this method, we glycine is converted to serine, also showed that – as expected – limiting of p53 stability and a couple of years ago Associate Scientist were able to demonstrate that increasing depleting the one carbon pool dietary serine would not completely prevent described the ability of a deubiquitylating Eric Cheung1 Our past work showing that many tumour cells exogenous glycine levels in cells deprived of and inhibiting proliferation. tumour growth. We are therefore testing enzyme (DUB), USP42 to drive the rapid have a very high demand for exogenous serine serine led to an increased conversion of additional interventions that might cooperate stabilisation of p53 in response to stress. By Research Scientists has led us to explore in more detail why cancer endogenous glycine to serine – a process that with serine depletion to kill, rather than inhibit removing ubiquitin from p53, USP42 accelerates Evrim Gurpinar Andreas Hock cells develop this dependency, and whether this would deplete the one carbon cycle (Fig. 1). To the proliferation of cancer cells. Our previous the activation of p53, although we showed that Flore Kruiswijk can be used for diagnosis or therapy. Serine and confirm our model that glycine inhibited cell studies showed that the adaptation to low USP42 is not essential for the induction of p53. Christiaan Labuschange1 glycine can be interconverted by the enzymes growth by exhausting the pool of one-carbon exogenous serine levels involves a switch to de However, during the course of these studies we Timothy Humpton1 SHMT1 (in the cytosol) and SHMT2 (in the units, we rescued the growth of glycine-only novo serine synthesis, which requires an also noted that USP42 could regulate Oliver Maddocks1 mitochondria) and it has been thought that cells fed cells by adding formate. By following the fate increased in oxidative phosphorylation and transcription independently of p53, Inbal Mor1 can use serine and glycine interchangeably. For of labelled glycine and formate under these enhanced anti-oxidant support. This work demonstrating that USP42 has p53- Associate Clinical Scientist this reason, much of our previous work used conditions we verified that the use of glycine for suggests that combining serine depletion with independent functions. This was not Patricia Roxburgh conditions that starved cells of both serine and nucleotide synthesis was blocked at a step prior inhibitors of oxidative phosphorylation or unexpected - as with other protein modification glycine. Over the past year, however, we have to the requirement for one-carbon units, and increased oxidative stress may lead to a more systems, the potential number of target proteins Scientific Officer discovered that this paradigm is not true in many that this was fully relieved by the addition of durable therapeutic response. exceeds the number of DUBs (around 100 in Robert Ludwig cancer cell lines, and we showed that formate as a one-carbon donor. Our results human). It is clear, therefore, that each DUB is Graduate Student exogenous glycine, in the absence of serine, suggest that many cancers may depend on We have shown in the past that the mutant p53 likely to target many different proteins. In a Pearl Lee2 cannot support cancer cell proliferation. Using exogenous serine for growth but could be proteins expressed in many cancers can continuation of our analysis of USP42 function, Koji Nomura metabolic flux analysis, we showed that cancer inhibited by increased concentrations of glycine. contribute to the invasive and metastatic we identified mono-ubiquitylated histone H2B cells selectively consume exogenous serine, We suggest that the elevation of the activity of behaviour of tumour cells, and that this is related as a target for deubiquitylation by USP42. This is 1 ERC and under fully fed conditions excrete glycine. the glycine cleavage system, previously to the ability of some of these mutant p53s to an interesting observation because it is known 2 MRC The serine that is taken up is converted to described in small cell lung cancers, may play a bind and inhibit the transcriptional activity of that the dynamic interchange and balance glycine, a process that provides a one-carbon role in degrading excess glycine that might p53 family members such as TAp63 and TAp73. between ubiquitylation and deubiquitylation of unit to support the production of nucleotides, otherwise accumulate and cause the growth Importantly, wild type p53 does not bind to its histones is critical for the regulation of as well as other important metabolic pathways inhibitory effects that we protein relatives and the interaction of mutant transcription. USP42 co-localised with RNA pol (Fig. 1). However, tracing the fate of glycine in have described. p53 with p63/p73 is dependent on the type of II in nuclear foci, bound to histone H2B and cells starved of serine indicated that while the mutations in the p53 protein. In general, deubiquitylated H2B. Depletion of USP42 glycine could be taken up and used for the We are now considering how to use our results mutations in p53 that affect protein increased H2B ubiquitylation at a model synthesis of glutathione (GSH), the exogenous for cancer detection or treatment. Our work and conformation (like p53R175H) show strong promoter, and decreased both basal and glycine was unable to support nucleotide that of others has shown that many cancer cells binding to p63 and p73, while p53 mutants that induced transcription from a number of synthesis. The ability of glycine to feed the one avidly consume serine, and if this characteristic only mildly affect the conformation (like promoters. These results showed that USP42 carbon cycle depends on the activity of the is confirmed in tumours in vivo, it may be p53R273H) bind less well. However, there are regulates transcription independently of p53 glycine cleavage system (GCS) to generate possible to use radioactive serine to label and many other protein-binding partners for the p53 through the control of histone ubiquitination, Me-THF in mitochondria. However, depletion of image cancers – similarly to the PET scanning family, including MDM2, a ubiquitin ligase that and suggest that USP42 may be an important the GCS did not affect cell growth, suggesting methods presently in clinical practice that targets p53 for ubiquitin dependent component of this fundamental level of that this pathway was not functional in tumour measure glucose uptake. In initial studies we are degradation. We found that both mutant p53 transcriptional activity. cells grown in culture. Interestingly, we also testing whether tumours differentially take up and p73 bind MDM2 very efficiently – although Publications listed on page 86

22 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR SUPPRESSION 23 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 Shehab Ismail - Structural Biology of Cilia 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 25 TUMOUR CELL MIGRATION www.beatson.gla.ac.uk/research/kurt_anderson

Our work is focused on development of imaging approaches Ewan McGhee (BAIR) on a fellowship funded by harmonic generation (SHG) can be used in INSPIRE to develop an adaptive optics module place of two-photon excited fluorescence to study the cellular and molecular dynamics of metastasis for use on one of our LaVision TRIM scopes. The as a signal for mirror optimisation. This is in vitro and in vivo. Metastasis is linked to mortality in most module they developed was based on a advantageous because unlike fluorescence, deformable mirror placed in the OPO the second harmonic signal does not photo- epithelial cancers. Metastatic invasion is challenging to study illumination pathway and imaged via a 4f system bleach over time. As a result, signal because it occurs randomly over large scales of time and into the back-focal plane of the microscope improvements of between two and four fold objective. The mirror shape was determined were achieved in a variety of samples, including space, and sensitively depends on features of the local tumour using a random search algorithm that was organotypic cultures, zebrafish embryos, and microenvironment. Our goal is to develop mechanistic optimised on the basis of signal intensity from freshly excised mouse skin and intestinal tissue the sample. Significantly, we found that mirror (Fig.2). This work is being continued in Group Leader readouts of cell migration and apply them to mouse cancer Kurt Anderson shapes optimised using one sample were collaboration with LaVision Biotec using their models including pancreatic ductal adenocarcinoma, transferrable to others, meaning that the lengthy new adaptive optics module, which has been FRMS optimisation procedure does not need to be installed on our new TRIM scope. melanoma and breast. performed prior to each imaging session. Also, Research Scientists the signal derived from collagen second Publications listed on page 78 Kirsty Martin1 Ross McQueenie We were the first group to apply fluorescence Biosensor development for drug discovery recovery after photo-bleaching (FRAP) in mice, This year we began a joint project Owen Graduate Students Figure 1 Orchi Anannya to study the dynamics of the cell-adhesion Sansom’s group and Novartis to study the role The Rac-Raichu FRET probe was Zahra Erami Rud Majani tumour suppressor E-cadherin in tumour cell of GEFs in pancreatic and colon cancer. To expressed under the control of Max Nobis migration. More recently, we have accomplish this work Novartis have funded a Villin-Cre in the crypts of mouse Meg Pajak2 demonstrated the first in vivo use of postdoc (Kirsty Martin) to develop the use of intestine. Intensity imaging fluorescence lifetime imaging (FLIM) to study FRET biosensors to probe the activation of Rac revealed membrane localisation 1 Novartis that delineated cell-cell 2 SINAPSE the activation and response to therapy of the and Ral GTPases. Kirsty started in June, and has boundaries (A and D). small GTPase Rho and the non-receptor begun to characterise optimal FRET pairs for Fluorescence lifetime imaging tyrosine kinase Src during mutant p53 driven fluorescence lifetime imaging of FRET. A (FLIM) revealed a basal activation invasion of pancreatic cancer cells. significant benefit of the biosensor approach is level having an average lifetime that the same assay will be directly translated of approximately 1.9 ns (B and C). Ex vivo treatment of tissue with Nuclear imaging from cell-based assays into preclinical studies. PMA resulted in significant (p < We continued to develop the use of preclinical We also contributed to the characterisation of a 0.05) shortening of the imaging in collaboration with colleagues from mouse capable of tissue specific expression of fluorescence lifetime to 1.7 ns NHS Greater Glasgow and Clyde, including the Rac-Raichu probe. The probe was readily due to FRET, indicating activation Gerry Gillen and the clinical team (Glasgow PET expressed and imaged in a variety of tissues of the probe. (Taken from Johnsson et al., Cell Rep 2014; 6: Centre), Jonathan Owen (Gartnavel Cyclotron) including the intestine, pancreas and mammary 1153) and Sally Pimlott (Radiopharmaceutical tissue. Activation with PMA demonstrated the Dispensary). In addition to FDG imaging, two utility of this mouse model for drug new probes were imaged this year: the development studies (Fig. 1). Figure 2 proliferation marker 18F-3’-Fluoro-3’-deoxy-L- Images of mouse skin showing approximately two fold gain in Thymidine (18FLT) and the metabolic marker Development of adaptive optics for deep signal resulting from the use of 18 18 Fluoro-acetate. FLT was synthesised tissue multiphoton imaging adaptive optics. SHG from manually and used to measure the response of Adaptive optics has the potential to increase collagen I (green) and KRAS-PTEN pancreatic tumours to inhibition of signal intensity and spatial resolution by fluorescence from Quantum the mTOR pathway by rapamycin. Work is sharpening the focus of a laser beam used for Dots (red) were imaged in mouse skin using either a flat (left) or currently underway at the Gartnavel Cyclotron multiphoton excitation deep within tissue. This optimised (right) mirror shape. to begin automated FLT synthesis, with approach can be use to correct for aberrations preclinical demand serving as a test run for in focus introduced by both the optics of the clinical production. We also embarked on a imaging system and refractive index variations collaborative project with David O’Hagan of the sample. In collaboration with Amanda (University of St Andrews) to produce 18Fluoro- Wright and Keith Mathieson (Institute of acetate, which was used by Eyal Gottlieb’s lab to Photonics, University of Strathclyde), Caroline investigate metabolic changes in colon cancer. Müllenbroich spent six months working with

26 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE TUMOUR CELL MIGRATION 27 survival. These data were validated in a second TRANSLATIONAL CANCER THERAPEUTICS group of patients in which low PTEN expression www.beatson.gla.ac.uk/jeff_evans was again associated with significantly poorer survival. In human tumours, approximately 20% of cases demonstrated low PTEN expression and have a gene expression signature that overlaps with murine KRAS PTEN tumours, suggesting that this could be applicable to enrich patient selection in subsequent human trials.

Optimising therapy in localised Current strategies in the development of new therapies for pancreatic cancer Two of the key issues in optimising therapy malignant disease are based on exploiting our increasing of localised pancreatic cancer are (a) inhibition understanding of the molecular and cellular basis of cancer of metastases and (b) local control of inoperable disease. development and progression. One aim of our work is to determine if potentially anti-invasive agents have anti- Work in our group is aimed at developing novel Exploiting tumour vulnerabilities: Figure 1 Manchester), Andrew Biankin and Sean migratory and hence anti-invasive and/or laboratory models that will allow us to implications for therapeutic intervention in Low PTEN and expression of a Grimmond (University of Glasgow), and the anti-metastatic properties. Our initial studies understand the biological function of key advanced pancreatic cancer low PTEN-associated signature G12D/+ Group Leader CRUK Clinical Trials Unit, Glasgow. This study used the Pdx1-Cre, Z/EGFP, L SL-Kras , tumour suppressor genes and oncogenes in We have developed a number of novel models predicts poor survival in human will consist of a series of parallel, early phase, L SL-Trp53 R172H/+ (KPC) model and cell lines Jeff Evans vivo in both normal tissues and tumours, and with a range of genetic backgrounds in PDAC. (A) Kaplan–Meier analysis efficacy signal-seeking studies in which patients developed from these mice. We demonstrated that will allow us to identify and characterise the collaboration with Owen Sansom’s group, of Glasgow cohort showing FRCP Edin cases with low PTEN expression will be recruited into multiple treatment arms of that dasatinib, an inhibitor of Src family kinases, signalling pathways that are deregulated at the including those with targeted deletion of Pten, have poorer outcomes. (B) inhibited cancer cell migration and invasion in ARF specific agents based on their molecular profile. Associate Scientist early stages of pancreatic cancer, and during the Apc, c-Myc or p19 . We are using these models Multivariate analysis showing low Critical to these approaches will be identifying vitro, and inhibited the development of Jennifer Morton development and progression of the invasive to help us understand the biological function of PTEN expression is an potential genotype-specific biomarker metastases in vivo. An international, multicentre and metastatic phenotype, and that are key tumour suppressor genes and oncogenes independent predictor of Research Scientist signatures in murine models and confirming the clinical trial based on this work has now potential therapeutic targets in advanced in vivo in both normal tissues and tumours, and survival. (C) Kaplan–Meier Amy Au1 analysis of Australian clinical relevance of these in human tissue completed accrual (Evans, global Chief 1 disease. Using these models, we will determine to allow us to identify and characterise the Mona Foth confirmatory cohort. (D) microarrays, and developing robust assays for Investigator), incorporating freedom from how potential anticancer agents might best be signalling pathways that are deregulated at Principal component analysis of patient selection to select or enrich the clinical distant metastases as a novel exploratory Scientific Officer evaluated in subsequent clinical trials. the early stages of pancreatic cancer, during gene expression data generated trial population. endpoint in patients with inoperable, locally Saadia Karim the development and progression of the from tumours in mice. (E) Heat advanced (but without metastases) disease. Infiltrating ductal carcinoma of the pancreas invasive and metastatic phenotype, and in map showing the PTEN- 1 University of Glasgow deficient signature could be used This approach is exemplified by our (PDAC) is the fifth commonest cancer in the UK, advanced disease. to delineate three groups of observations that PTEN loss leads to Having used this approach to refine the clinical and is predicted to become the second patients. Black indicates low or acceleration of KrasG12D-driven PDAC in evaluation of existing anticancer agents, we are commonest cause of cancer-related deaths by We are using these murine models to explore negative, while grey indicates murine models and these tumours have high now using principal component analysis, in the end of this decade. Aggressive invasion and tumour vulnerability in vivo to specific targeted high or positive values. (F) levels of mammalian target of rapamycin collaboration with Owen Sansom and Andrew early metastases are characteristic of the agents. In addition to conventional response Kaplan–Meier analysis showing of Glasgow cohort cases (mTOR) signalling. We compared response to Biankin (University of Glasgow), to define a disease, such that 90% of patients have assessments and survival studies, we also delineated on the basis of gene mTOR inhibition in this model with the response functionally relevant signature associated with surgically unresectable disease at the time of monitor therapeutic activity using a PET/SPECT/ expression of low PTEN- in another established model of pancreatic survival in resected PDAC in order to understand diagnosis. Overall survival remains poor for both CT preclinical imaging system (collaboration associated signature. Cases with cancer, KRAS P53. We found that tumours in the key pathways that drive poor survival and resectable and advanced disease using with Kurt Anderson and Gaurav Malviya), high expression of signature (red) KRAS PTEN mice exhibit a remarkable identify targets for the development of novel conventional therapies, and has only improved enabling us to use clinically relevant imaging have significantly decreased survival compared to those with dependence on mTOR signalling. In these anti-invasive agents. marginally over the last few decades with a probes to assess therapeutic efficacy with medium (green) or low tumours, mTOR inhibition leads to proliferative preponderance of negative clinical studies using regard to tumour metabolism and tumour expression (blue). (G) Heat map arrest and even tumour regression. Importantly, Optimal local control remains an important current trial designs. Preclinical modelling has proliferation, and which can then be showing validation of the pancreatic tumours driven by activated KRAS clinical issue in patients with non-metastatic invariably failed to deliver robust results in incorporated into clinical protocols. Our initial PTEN-deficient signature used to and mutant p53 did not respond to treatment. disease, particularly in those with ‘borderline’ predicting success or in reducing drug attrition focus has been on targeting pathways that are delineate three groups of patients. (H) Kaplan–Meier operable disease, and in whom chemo- in pivotal clinical trials. Consequently, novel (a) implicated in PTEN deletion (PI3K / AKT, curves showing difference of We also assessed whether there was a subset of radiation is frequently used. Inhibitors of approaches are urgently required. mTORC 1 and 2); (b) downstream of mutant overall survival between three human pancreatic cancer patients who may poly(ADP-ribose) polymerase (PARP) may have KRAS (e.g. effector pathways); and (c) intra- and groups of patients identified by respond to mTOR inhibition. PTEN expression radio-sensitising effects and a phase I/II study of Our work aims to develop therapeutic peri-tumoural inflammatory pathways that the PTEN-deficient signature in was determined by immunohistochemistry on a olaparib in combination with chemo-radiation interventions for advanced pancreatic cancer by are implicated in tumour initiation and the confirmatory cohort. tissue microarray of resected human pancreatic in locally advanced pancreatic cancer will exploiting tumour vulnerabilities in preclinical tumour progression. tumour specimens, and patients were divided commence soon (Experimental Cancer models with specific genetic backgrounds, and into groups of low and high expression Medicine Centres-Industry Alliance). to optimise therapy of localised pancreatic We will exploit these studies to inform a quantified using a histoscore method. Low cancer through inhibition of metastases and UK-wide MAMS (multi-arm molecular stratified) PTEN expression was associated with Publications listed on page 79 through optimising control of localised clinical trial that is currently in development in significantly poorer survival in these patients. inoperable disease. collaboration with Juan Valle (University of Furthermore, by multivariate analysis, low PTEN expression was an independent predictor of

28 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE TRANSLATIONAL CANCER THERAPEUTICS 29 Regulators of the Arp2/3 complex in We have also used chemotaxis chambers of our CELL MIGRATION AND CHEMOTAXIS actin dynamics own design to show that melanoma cells are www.beatson.gla.ac.uk/robert_insall Actin drives nearly all cell movement, and the exquisitely chemotactically sensitive. They can principal driver of actin is an assembly called the navigate up a gradient of serum with Arp2/3 complex. When turned on, the Arp2/3 unprecedented accuracy, irrespective of their complex causes new actin filaments to form stage – early melanomas are slower but still and push against the membranes inside and at highly chemotactic. We are now working on the the leading edge of cells. We are particularly molecular details of the attractant in serum and interested in the family of proteins that turns on the chemotactic receptors that detect it. the Arp2/3 complex. The most interesting part of a melanoma cell’s One such regulator is SCAR/WAVE, which is a response is that we find they make their own Crawling movement is fundamental to the behaviour of most fundamentally important regulator of cell chemotactic gradients. LPA - which appears to movement. Mutants in a variety of species show be present at substantial levels in the tissue eukaryotic cells. In most tissues it is suppressed, so cells remain that it is required whenever cells need to make surrounding tumours - is a strong attractant for within the correct organs. However, when tumours large actin-based structures such as all the melanoma cells we have observed. But lamellipods; without SCAR/WAVE such melanoma cells also break down LPA. This leads metastasise, the regulation of cell movement goes wrong - structures are either small and malformed, or to a self-generated gradient, in which cells cancer cells invade other tissues, and spread through the completely absent. It is found as part of a move out of tumours in response to gradients five-membered complex with the Rac-binding they are themselves creating. Thus tumours blood and lymph systems to form secondary tumours. This protein PIR121, Nap1, Abi and HSPC300. appear to need no external drivers to steer spreading behaviour is one of the most feared features of Without the other members of the complex, metastasis – they do it themselves. Group Leader cancer and a principal driver of death in patients. Despite this, SCAR is rapidly removed from the cell. The Robert Insall prevailing view in the field is that all these We are collaborating with the Mathematics we do not understand what makes cells move or what steers proteins act simultaneously as a huge, Departments of the University of Strathclyde FRSE homogenous complex that couples Rac and and the University of Glasgow to make different their direction. Our group aims to improve understanding lipid signalling to actin polymerisation. However, computational models representing moving Research Scientists this view seems very simplistic in view of the size cells. Our models already faithfully mimic some Jan Ohotski using all means at our disposal, for example in vivo models, of the complex and its dynamic behaviour. aspects of the movement of Dictyostelium cells. Olivia Susanto cancer cells, model organisms and computational simulations. Luke Tweedy We are now using the model to test our We apply a wide range of techniques, from genetics through Our experiments are currently focused on predictions about the underlying mechanisms Scientific Officer identifying the activators and other proteins that of chemotaxis, and the proteins that are Peter Thomason biochemistry and technical microscopy to quantitative analysis regulate each component of the complex. involved. We are showing that chemotaxis is

Graduate Students of microscope movies and computational modelling. SCAR and the other complex members are mostly likely mediated by several dissimilar Clelia Amato phosphorylated at multiple sites but the mechanisms acting in parallel, including José Batista1 biological significance of these regulated pseudopod growth, pseudopod Yvette Koh We are particularly interested in two related but remains very poorly understood. We are phosphorylations is not understood. We have retraction and the control of adhesion. We can questions. The first is chemotaxis, in which trying to understand cell movement – what shown that control of SCAR phosphorylation is also determine which components can safely 1 FCT external signals orient and attract cells, and drives it and most importantly how it is steered. centrally important – nearly all the cellular SCAR be ignored, which is increasingly important which is increasingly seen as a fundamental is heavily phosphorylated but a rare – hundreds of genes are newly associated with cause of metastasis. The second is the Most mammalian cells use pseudopods made dephosphorylated form seems to be particularly motility and invasion every year so we urgently regulation of the Arp2/3 complex, an assembly of polymerised actin to power migration. Our important. It is also very active in extending need a mechanism to determine which are the of proteins that promotes movement by driving current research focuses on the proteins and pseudopods, and very unstable, explaining its most important. Our theory of self-generated the formation of actin microfilaments. About pathways that control these pseudopods. We rarity. We are now seeking the phosphatases. gradients implies that cells behave in a similar half of the lab has a genetic focus, which varies use three approaches. For genetic studies we We have also shown – very unexpectedly – that way to herds of animals in the wild. We are in detail in response to the mutant phenotypes use Dictyostelium, taking advantage of its ease nearly all the same signals regulate the therefore also collaborating with mathematical we see. However, our strategy is always based of manipulation, prominent cell movement and localisation of SCAR and its relative WASP. We ecologists at the University of Glasgow to around chemotaxis and actin. chemotaxis. To apply our knowledge to cancer, are now seeking to understand what those determine whether this comparison can yield we use a range of melanoma cells cultured from signals are, and how they connect to upstream useful predictions about cancer cell behaviour. Cell movement is a central part of biology, from tumours with different degrees of metastasis, signalling molecules such as receptors and conception to death. Embryos form their and actual tumours from mouse models and, G-proteins. Publications listed on page 81 complexity and shape from the movement of when possible, from fresh patient tissue. We also layers of cells as well as the migration of develop computational models of single cells in Mechanisms underlying chemotaxis: individual cells through tissues. Adult wound collaboration with the Mathematics Pseudopods and self-generated gradients healing and responses to infection require skin Department, University of Strathclyde, and of Chemotaxis, migration towards chemical and immune cells to migrate to where they are populations of moving cells with the Institute of signals, is emerging as a major driver of tumour needed. Metastasis, one of the most feared Biodiversity Animal Health and Comparative metastasis. We have shown that chemotaxis in features of cancer, is caused when cells migrate Medicine, University of Glasgow. In the long Dictyostelium cells works by a different out from a tumour into the blood, lymph or term, we will work on anything that will help us mechanism than that which is usually described. other tissues. Chemotaxis, the connection to understand the conserved and fundamental Pseudopods are constantly generated in between chemical signals outside the cell and mechanisms that drive cell movement. random directions, then the ones that point in its movement, is important in these processes the best directions are selected and maintained.

30 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE CELL MIGRATION AND CHEMOTAXIS 31 STRUCTURAL BIOLOGY OF CILIA www.beatson.gla.ac.uk/shehab_ismail

The primary cilium is a microtubule-based sensory organelle that the solubilising function of GSFs is regulated Ciliary access for integral membrane proteins by the small G proteins Arl2 and Arl3 in a GTP- and large soluble proteins that is present on almost all cell types in the body. While dependent manner (Ismail et al., 2011; Ismail et A diffusion barrier regulates access of integral lymphocytes lack cilia, due to functional and structural al., 2013). Currently, my group is trying to answer membrane proteins and large soluble proteins the question of how GSFs selectively target to cilia. A septin ring, a protein network that similarities, the immune synapse can be considered as a the now soluble cargo to different destinations resides in the ciliary transition zone, and the special type of primary cilium. The assembly of cilia is tightly in the cell. transition fibres connecting the basal body to the ciliary membrane are believed to control synchronised with the cell cycle and coordinates several Using biochemical studies, we show that Arl2, access to cilia and be involved in forming the signalling pathways such as the Wnt and Hedgehog localised outside cilia, releases the cargoes ciliary diffusion barrier (Fig. 1). The protein destined for the outside of cilia, while Arl3, network in the transition zone is thought to be Group Leader pathways, both of which are therapeutic targets for cancer. Shehab Ismail localised within cilia, releases those cargoes part of the ciliary diffusion barrier and to have a Furthermore, the immune synapse plays a central role in destined for the cilia itself. gating function. Many of the network members Scientific Officer are involved in ciliopathies. The exact identity of Michael McIlwraith tumour immunology. Concentrating signalling proteins and We are currently investigating the structural the ciliary gate, the molecular mechanism of its receptors inside the cilium is key to its function. Nevertheless basis of the interaction of several lipid-modified function and its structural, biochemical and cargoes with GSFs in order to understand the dynamic properties remain elusive. In our the cilium, unlike other organelles, is not separated from the selective release of cargoes by Arl2 or Arl3. group, we are working on obtaining atomic level cell body by a membrane barrier and the molecular Furthermore, we are studying the effect of these details of the ciliary gate and its biochemical interactions on the functions of lipid-modified characterisation by reconstituting multimeric mechanism for regulating ciliary targeting and access of proteins. Finally, we will vigorously test the protein complexes of the ciliary transition zone relevant proteins to cilia is elusive. structural and biochemical data in cells. and using X-ray crystallography. Publications listed on page 81 Our group aims to unravel the regulation of to their cellular destination in complex with ciliary access, for membrane associated, integral GDI-like solubilising factors (GSFs). Three Figure 2 membrane and large soluble proteins (Fig. 1), by homologous GSFs, PDE (delta subunit of Ciliary and non-ciliary cargo using an interdisciplinary approach where we phosphodiesterase), UNC119a and UNC119b, release from UNC119a via Arl2 combine structural biology, biochemistry and contain hydrophobic pockets that and Arl3 GTP. a) Fluorescence cell biology. accommodate and bury lipid moieties of polarisation measurement post-translationally modified, membrane- where UNC119a is added to fluorescently labelled, Ciliary access of membrane associated associated proteins and hence function as myristoylated ciliary or non- proteins solubilising factors (Hanzal-Bayer et al., 2002; ciliary cargo, as indicated, Several lipid-modified, myristoylated and Ismail et al., 2011; Wright et al., 2011; Zhang et al., followed by the addition of Arl2 prenylated proteins are solubilised and targeted 2011). During my postdoctoral work, I showed or Arl3 GTP, as indicated. b) Cartoon representation showing our model of the release of Figure 1 lipid-modified cargo inside or A cartoon representation of a outside the cilium by Arl3GTP or cilium showing the different Arl2GTP, respectively. types of protein cargoes, as indicated, sorted to the cilium.

32 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE STRUCTURAL BIOLOGY OF CILIA 33 compounds in collaboration with Martin MIGRATION, INVASION AND METASTASIS Tubulin Cortactin merged Drysdale and Drug Discovery. www.beatson.gla.ac.uk/laura_machesky Role of actin regulatory proteins in melanoblast migration and melanoma We previously showed that loss of Rac1 causes major defects in melanoblast migration and proliferation during development (Li et al., Dev Cell 2011; 21: 722) and now we are studying the roles of RhoA and Cdc42 in melanoblasts with Figure 1 We are also studying the role of fascin-1 in PDAC PhD student Emma Woodham and Ben Tyrrell, The leading edge of an invading progression, a project initiated by former together with Cord Brakebusch (BRIC, University melanoma cell is shown with student Ang Li (now at Rockefeller University in of Copenhagen, Denmark). Emma has found a Cancer metastasis requires cells to break away from the microtubules in red and the major role for Cdc42 in melanoblast migration primary tumour and to survive in a variety of environments invadopodia protein cortactin in New York) and now continued by Amelie Juin, green. Microtubules play an Hayley Morris and Loic Fort in collaboration with and proliferation and is currently working with before settling into a new site. We aim to gain insights into important role in invading Jennifer Morton and Owen Sansom. This year, cultured cells to unravel the molecular pseudopods and coordinate with we published that fascin-1 is upregulated in mechanisms. With Ang Li, we published this year the actin cytoskeleton to deliver mechanisms of metastatic spread by determining the roles of human PDAC and correlates with poor that the Rac1 activator protein pREX-1 also has receptors and proteases to aid key actin cytoskeletal proteins, such as the actin filament matrix remodeling. Photo Credit: outcome and time to recurrence of disease (Li et roles independent of Rac1 in melanoblasts Iben Veland al., Gastroenterology 2014; 146: 1386). We found (Lindsay et al., J Invest Dermatol 2015; 135: 314). nucleation machinery and the bundling protein fascin-1, in that fascin is a target of the epithelial to With former postdoc Yafeng Ma, we also cancer cell invasive and migratory behaviour. The actin mesenchymal transition, which allows tumour published that fascin is expressed in some Group Leader cytoskeleton is important not only for cell strength and cells to break away from their neighbours. melanomas but is not tightly correlated with Laura Surprisingly, fascin-1 has a key role in early stage or progression as it is in many epithelial migratory capacity but also for adhesion-dependent survival, tumour formation as well as metastatic cancers (Ma et al., Melanoma Res 2014, in press). Machesky dissemination. Fascin mediates metastasis to the A new postdoc, Karthic Swaminathan joined the membrane trafficking and establishment of polarity. We aim to FRSE peritoneal cavity via the formation of actin-rich team this year and he will study the role of the understand how various actin regulators control these filopodia that enable cells to intercalate and major actin nucleation promoting complex Research Scientists migrate through mesothelial cell layers and Scar/WAVE in melanoblast migration and Amelie Juin processes and thus contribute to tumour initiation, growth and seed a new tumour. Fascin also contributes to melanoma metastasis. He and Loic Fort are also Karthic Swaminathan metastasis as well as to fundamental mechanisms of liver metastasis. This work complements our setting up a model to study melanoma Ben Tyrrell ongoing efforts to develop fascin-1 inhibitor metastasis in collaboration with Jim Bear (UNC, Iben Veland1 mammalian development. compounds together with Martin Drysdale’s USA) where we will use intravital imaging to Scientific Officer Drug Discovery team. We also have a new track cells escaping from tumours. Heather Spence Role of actin nucleating proteins in cell microtubule network connects to actin to affect project funded by the Pancreatic Cancer Publications listed on page 82 migration, invasion and membrane trafficking polarised cell invasion (Fig. 1). PhD student Loic Research Fund to further probe the mechanism Clinical Research Fellows of fascin involvement in PDAC and to test Hayley Morris2 The Arp2/3 complex is the major inducer of Fort is working in collaboration with Jose Batista actin filaments in response to extracellular in Robert Insall’s group to discover new Graduate Students signals. The Wiskott-Aldrich Syndrome Protein regulators of the Scar/WAVE complex and study Figure 2 Loic Fort family proteins (including WASP/N-WASP, Scar/ their role in migration and invasion. Left- Pancreatic cancer cell Emma Woodham spheroids invading into collagen WAVE, WASH, WHAMM and JMY) transmit gel (green = actin, blue = DNA). 1 University of Copenhagen/ signals to the Arp2/3 complex to trigger actin Role of actin regulatory proteins in colorectal Right- Pancreatic cancer cells Danish Research Council assembly. Each of these proteins is regulated and pancreatic cancer invading into Matrigel (red = 2 MRC Clinical Research differently and one of our aims is to understand This year, clinical research fellow Hayley Morris actin, green = cortactin). Fellowship the mechanisms of regulation and the continued her study of the role of N-WASP in Photo Credit: Amelie Juin involvement of these proteins in invasion and colorectal cancer (in collaboration with Owen metastasis of cancer as well as their normal Sansom) and with postdoc Amelie Juin on the cellular function. WASP family proteins regulate role of N-WASP in pancreatic ductal actin assembly in multiple essential and adenocarcinoma (PDAC). High levels of pathological cellular processes, such as N-WASP have recently been correlated with endocytic trafficking, protrusion of lamellipodia poor outcome in human patient PDAC (Guo et Figure 3 and filopodia, cell division and assembly of al., World J Surg 2014; 38: 2126) pointing to it as Melanoblast cells migrating in invasive structures such as podosomes and a potentially interesting new target for PDAC. We epidermis (green = GFP- invadopodia. Postdoc Ben Tyrrell is studying the will determine whether the role of N-WASP in melanoblasts, blue = DNA). Left: role of the WASH protein complex in the invadopodia translates into differences in WT = wild type control, Right: Cdc42 f/f = deleted for Cdc42. production of actin networks on endocytic tumour formation, progression and spread. Photo Credit: Emma Woodham vesicles to regulate cell signalling and motility in Thus far, we find a strong role of N-WASP in three-dimensional matrices. New postdoc Iben invasion and metastasis and we are working to Veland is studying the role of WASH proteins in discover the mechanisms by which the actin invasive migration and the delivery of matrix cytoskeleton confers invasiveness and metalloproteases and receptors into invasive metastatic capability on pancreatic cancer pseudopods. She is also interested in how the cells (Fig. 2).

34 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE MIGRATION, INVASION AND METASTASIS 35 Met to influence their invasive behaviour. Indeed, we transfer RNA (tRNAi ), are up/downregulated INTEGRIN CELL BIOLOGY have shown that p53 null cells alter their RCP- and transgenic mice that express additional dependent integrin trafficking and adopt an copies of the tRNA Met gene. www.beatson.gla.ac.uk/jim_norman i invasive migratory phenotype when exposed to exosomes from mutant p53 expressing cells. A series of experiments conducted using the Met These findings indicate that a small population tRNAi overexpressing mouse indicate that an of mutant p53 expressing cells are potentially important component of Pol III’s drive to able to influence the aggressiveness of other tumour progression is due to the action of Met less invasive cells in a systemic fashion, and increased tRNAi in the tumour stroma, not in indicates the need to look for ways to oppose the tumour cells themselves. These data Rab35-dependent exosome release in indicate that a key role of Pol III and its product Met cancer therapy. tRNAi is to support the synthesis and secretion Integrins are cell surface receptors that engage the extracellular of stromal collagen II – an event that promotes The role of translation initiation in assembly of an extracellular matrix (ECM), which matrix and help cells to move. Cancer cells use integrins in extracellular matrix synthesis during supports tumour progression in vivo. We are order to migrate away from primary tumours to form tumourigenesis currently investigating how altered levels of Met Several RNA polymerase III (Pol III)-associated tRNAi favour synthesis of collagen II-rich metastases. Like many other receptors, integrins are genes are increased in tumours and tumour pro-tumourigenic ECM, and how control of internalised (or endocytosed) from the cell surface into metastases. We have generated a number of translation initiation in stromal fibroblasts tools to determine how Pol III may contribute to may be a key factor in determining cancer endosomes. Once within endosomes, integrins can either be cancer progression – these include a panel of aggressiveness. sorted for degradation or can recycle to the cell surface via a cell lines in which Pol III and its principal Group Leader number of different routes. We have found that a range of products, including the initiator methionine Publications listed on page 83 Jim Norman drivers to cancer metastasis operate by altering the way in Figure 1 A Research Scientists EphA2 LMTK3 phosphorylates RCP to Jesica Diaz-Vera which integrins recycle to the plasma membrane. Moreover, drive EphA2 trafficking and Emmanuel Dornier we now know that components of the integrin recycling cell:cell repulsion.(A) Lemur Liane McGlynn tyrosine kinase-3 (LMTK3) is Elena Rainero1 EEs machinery influence clinical outcomes in patients with inactive and the Ephrin receptor, Peter van den Berghe EphA2 is internalised and returns Cell:cell junc*on pancreatic and breast cancer. We will continue to assemble a to the plasma membrane from Associate Clinical Scientist REs early endosomes (EEs). Under Iain MacPherson detailed molecular picture of integrin recycling and how this RCP these circumstances, stable Rab11 cell:cell junctions are Scientific Officer contributes to metastasis, and hope to reveal which are the maintained. (B) active LMTK3 Louise Mitchell most promising components of the pathway to target for phosphorylates Rab-coupling B protein (RCP) at Ser435 enabling Graduate Students cancer therapy. RCP to associate with Rab14. Cassie Clark EphA2 is then transported, under Cell: cell Nikki Heath control of Rab14, to recycling Nicholas Rabas EEs repulsion endosomes (REs) and returns to Elena Ruengeler RCP controls recycling of an Ephrin receptor agents to target LMTK3 kinase to oppose RCP the plasma membrane with to promote cell:cell repulsion and tumour phosphorylation and tumour cell dissemination. delayed kinetics. This process REs 1 West of Scotland Women’s cell dissemination LMTK3 promotes EphA2-dependent RCP Bowling Association Having characterised the role of the Rab11 A new role for exosomes in transferring cell:cell repulsion leading to cell Ser435 Rab14 Fellowship effector, Rab-coupling protein (RCP) in integrin mutant p53-driven invasive phenotype scattering, and dissemination P recycling, we have been using proteomic and between cells and metastasis of tumour cells. phospho-proteomic approaches to determine We have previously shown that mutants of the Figure 2 whether other receptors might be cargoes of tumour suppressor p53 can promote cancer Exosome-­‐mediated Intercellular transfer of mutant Intercellular transfer the RCP pathway. We have identified a new invasion via a gain-of-function mechanism. p53’s invasive phenotype is Invasion trafficking pathway in which RCP, when Mutant p53s increase the invasive behaviour of mediated by exosomes. The phosphorylated at Ser435 by a kinase called cancer cells by upregulating RCP-dependent ‘donor’ cell depicted on the left expresses mutant p53, whereas LMTK3, is able to associate with a small GTPase, trafficking of integrins and receptor tyrosine α5β1 the ‘recipient’ cell on the right is Rab14 to control trafficking of an Ephrin kinases, such as EGFR1 and cMET. We have now Exosomes p53 null. In the ‘donor’ cell, Rab35 EGFR1 receptor, EphA2. Importantly, RCP must be found that the ability of mutant p53 to + mutant p53 activates a Rab35- EEs + RCP phosphorylated by LMTK3 in order for EphA2 to upregulate integrin trafficking and drive invasive dependent process that leads to exosome release. Exosomes may be capable of mediating the cell:cell repulsion migration is not cell autonomous, and mutant MVB REs then be transferred to ‘recipient’ events that drive tumour cells away from one p53-expressing cells can communicate these EEs cells. p53 null recipient cells another during cancer invasion. Consistently, pro-invasive capabilities to neighbouring cells respond to these exosomes by mp53 we have found that genetic disruption of either that do not express p53. This proceeds via a activating RCP-dependent α5β1 the EphA2 or the RCP genes in mice opposes mechanism in which mutant p53 drives Rab35- and EGFR1 recycling to promote dissemination and metastasis of pancreatic dependent production of exosomes. These tumour cell migration and invasion. EEs, early endosomes; adenocarcinomas. We are currently exosomes are then released from mutantp53- REs, recycling endosomes; MVB, Mutant p53-­‐expressing ‘Donor’ Cell P53-­‐null ‘Recipient’ Cell investigating the possibility of developing expressing cells and can interact with other cells multivesicular body.

36 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE INTEGRIN CELL BIOLOGY 37 MOLECULAR CELL BIOLOGY www.beatson.gla.ac.uk/michael_olson

Figure 1 A major function of the actin cytoskeleton is to provide the Lung cancer cells were fixed and structural underpinning that gives a cell shape and mechanical stained for their microtubule structures (red) and nuclear DNA strength. The actin cytoskeleton is dynamic, undergoing (blue). Cells treated with vehicle alone have well-formed constant rearrangement and reorganisation in response to microtubules, while cells treated with a LIMK inhibitor have shorter external factors including soluble factors and the physical and more chaotic microtubules. In addition, there is evidence of microenvironment. Alterations to the cytoskeletal architecture cells failing to undergo mitosis have significant consequences on the entire cell - such as successfully, which is associated with decreased proliferation. Group Leader morphology, cytokinesis, adhesion and motility – and also at Michael Olson the subcellular level. Research in our lab is focused on Research Scientists Katerina Mardilovich identifying key regulators of actin cytoskeleton dynamics that Nicola Rath contribute to processes that are important for cancer. In Mathieu Unbekandt1 particular, we have focused on kinase effectors that act Scientific Officer In collaboration with the Beatson’s Drug of Y27632, despite equivalent inhibition of MLC June Munro downstream of Rho GTPases. Through these efforts, we aim to Discovery Programme, funding was obtained phosphorylation. These results showed that Clinical Research Fellow uncover proteins that could be potential cancer drug targets. from the Medical Research Council and BDP5290 is a potent MRCK inhibitor with Jennifer Brown2 Worldwide Cancer Research to develop activity in cells, resulting in reduced MLC inhibitors for the MRCK proteins. Screening a phosphorylation, cell motility and tumour cell Graduate Students LIMK inhibitor development cancer by enabling the nuclear translocation of kinase-focused small molecule chemical library invasion. The discovery of this compound will Linda Julian resulted in the identification of compounds with enable further investigations into the biological Dominika Rudzka LIM kinases play central roles in Rho GTPase the androgen receptor in response to hormone regulation of the actin cytoskeleton by stimulation. In addition, we collaborated with inhibitory activity towards MRCK. Medicinal activities of MRCK proteins and their Visiting Students phosphorylating cofilin proteins (cofilin1, the Wellcome Trust Sanger Institute’s Genomics chemistry combined with in vitro enzyme contributions to cancer progression. Chloe Charalambous cofilin2, destrin/ADF) on Ser3 and inactivating of Drug Sensitivity in Cancer project to screen profiling led to the discovery of 4-chloro-1-(4- Lena Helbig their F-actin severing activity. The LIM kinase over 600 tumour cell lines for their sensitivity piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl] Publications listed on page 83 Lucia Ruiz Mata pyrazole-3-carboxamide (BDP5290) as a potent Adam McNee family consists of two members; LIM kinase 1 to two different LIMK inhibitors. These studies Haneela Qadir (LIMK1) and LIM kinase 2 (LIMK2), and their revealed that neuroblastoma was the most MRCK inhibitor. X-ray crystallography of the Sebastien Torke activation results from phosphorylation on sensitive to both inhibitors. Cell-based MRCKβ kinase domain in complex with specific residues in their kinase domains by Rho experiments revealed that an important BDP5290 revealed how this ligand interacts with 1 Worldwide Cancer GTPase–regulated kinases, including ROCK, mechanism of action of LIMK inhibitors in the nucleotide-binding pocket. BDP5290 Research PAK and MRCK. There are numerous ways that blocking proliferation is by inducing demonstrated marked selectivity for MRCKβ 2 CRUK Glasgow Centre Rho GTPase activation has been associated with mitotic defects. over ROCK1 or ROCK2 for inhibition of myosin II human cancers, particularly with progression light chain (MLC) phosphorylation in cells. While to invasive and metastatic stages. Therefore, MRCK inhibitor development BDP5290 was able to block MLC LIMKs make interesting prospective targets The myotonic dystrophy kinase-related CDC42- phosphorylation at both cytoplasmic actin for drug discovery. binding kinases, MRCKα and MRCKβ regulate stress fibres and peripheral cortical actin actin-myosin contractility and have been bundles, the ROCK selective inhibitor Y27632 CRT’s Discovery Laboratories undertook a high implicated in cancer metastasis. Along with the primarily reduced MLC phosphorylation on throughput screening and medicinal chemistry related ROCK1 and ROCK2 kinases, the MRCK stress fibres. BDP5290 was also more effective programme to develop potent and selective proteins initiate signalling events that lead to at reducing MDA MB 231 breast cancer cell LIMK inhibitors. Funding from the CRUK’s contractile force generation, which powers invasion through Matrigel than Y27632. Finally, Discovery Committee enabled us to undertake cancer cell motility and invasion. A potential the ability of human SCC12 squamous cell studies that aimed to identify the tumour types strategy for cancer therapy is to reduce carcinoma cells to invade a three-dimensional that were most sensitive to growth inhibition metastasis by blocking MRCK activity, either collagen matrix was strongly inhibited by 2 μM induced by LIMK inhibitors. We recently alone or in combination with ROCK inhibition. BDP5290 but not by the identical concentration reported that LIM kinases contribute to prostate

38 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE MOLECULAR CELL BIOLOGY 39 COLORECTAL CANCER AND WNT SIGNALLING www.beatson.gla.ac.uk/owen_sansom

Figure 1 A) Small Intes+ne Colon B) Colorectal cancer is the third most common cancer in the E-cadherin-β-catenin UK and the second most common cause of cancer mortality. complexes are increased in the colon compared to the small The focus of our group is to understand the early changes intestine. A) Complexes of β-catenin-E-cadherin (in red), associated with intestinal neoplasia in order to identify novel blue stain is DAPI nuclei in small intestine and colon. B) markers of the disease as well as new targets for therapy. Quantification of complexes in small intestine versus colon The key intestinal tumour suppressor is the APC gene that is (each colour spot represents a mutated in approximately 80% of sporadic cancers. Central different sample). C) β-catenin Group Leader immunohistochemistry to our work is the use of novel inducible models of intestinal showing 5 days post cre Owen Sansom induction, no nuclear β-catenin tumourigenesis that allow us to study the functions of in AhCreER Catnblox(ex3)/+ FRSE intestines apart from the base of AhCreER Catnblox(ex3)/+ specific tumour suppressor genes. the crypt (arrowed). When a ER lox(ex3)/+ C) AhCre Catnb Cdh1fl/+ Research Scientists single copy of E-cadherin is Patrizia Cammareri1 deleted there is now lots of Fatih Ceteci1 Elucidating why APC is so frequently mTORC1 is required for proliferation of nuclear β-catenin and much 2 William Faller mutated in colorectal cancer APC deficient cells larger intestinal crypts. Robert Fordham3 Rene Jackstadt A key unanswered question in colorectal cancer mTORC1 is a kinase that is required to Bryan Miller4 is why APC mutations are so frequent (~ 75%) coordinate proliferation and cell growth, and Claudio Murgia whilst activating mutations in β-catenin are rare has long been viewed as an attractive pathway Karen Pickering4 (5%) when both activate the WNT pathway. To for cancer therapy. Our work has shown that Figure 2 Inhibition of mTORC stops the Myriam Solar Abboud address this, we have compared the activation of mTORC1 is a common event David Vincent2 proliferation of APC deficient consequences of acute loss of APC and following loss of APC. This year, we have shown cells. A) Schematic of many of the phenotypes of APC loss, increasing cells and hope to come up with rationale Scientific Officers activation of β-catenin within the murine small that APC deficient cells are absolutely mechanism of how loss of Apc proliferation rates and accelerating combination therapies that will overcome this Tam Jamieson and large intestine. Compared to Apc loss, dependent on mTORC1 to proliferate (Faller et causes the activation of mTORC tumourigenesis. Importantly, APC deficient cells resistance mechanism and specifically target signalling. B) H&E stained Rachel Ridgway β-catenin activation took longer to lead to al., Nature 2015; 517: 497). Genetic inhibition or that also carry a Kras mutation are intrinsically cells carrying both of these mutations. 2 sections of intestines, wild type Andrew Campbell accumulation of β-catenin in the nucleus and use of rapamycin, a chemical inhibitor, Ee Hong Tan (WT) or APC deficient (APC). As resistant to mTORC1 and MEK inhibition. This acquisition of a crypt progenitor phenotype in completely removes the proliferative capacity of can be seen by the red bar, wild may help explain why later stage colorectal Publications listed on page 84 Graduate Students the small intestine and, more importantly, was APC deficient cells. This is associated with a type intestinal crypts are much cancer trials using mTORC1 inhibitors have not David Gay2 unable to transform the colon. We found that slowing of translational elongation and hence, smaller than APC deficient ones. worked. Mechanistically, Kras mutation causes 1 Treatment with rapamycin David Huels E-cadherin, which is known to interact with given the massive burst of transcriptionally the activation of mTORC1 targets in an 5 returns APC deficient crypts (APC Michael Hodder β-catenin and hold it at cell junctions hence activity of APC deficient cells, cells enter growth Pedro Salgueiro6 + rapamycin) to the same size as mTORC1-independent manner. We are now preventing nuclear accumulation, is expressed arrest (Fig. 2). Although this growth arrest is wild type ones. examining the pathways of resistance in these 1 EU PRIMES at much higher levels in the colon. Moreover, reversible, since rapamycin is a well-tolerated 2 ERC using a proximity ligation assay (PLA), we drug, it is possible that it would be beneficial 3 1851 Royal Commission discovered that there were many more for those patients that are predisposed to Research Fellowship β-catenin-E-cadherin complexes in the colon colorectal cancer. WT Apc Apc + 4 Novartis than in the small intestine (Fig. 1). Deletion of a Rapamycin 5 MRC single copy of E-cadherin (which alone has no Additional mutation of KRAS rewires APC 6 joint with A*STAR impact on intestinal homeostasis) in deficient cells to become resistant to combination with an activating β-catenin mTORC1 and MEK inhibition mutation, led to a rapid relocalisation of In colorectal cancer, KRAS is often co-mutated β-catenin to the nucleus and a crypt progenitor with APC (in ~40% of cases). These mutations phenotype. There was then rapid are activating and tumours carrying KRAS transformation of the murine colon. mutations have a poorer prognosis and are less Importantly, these differences were conserved likely to respond to therapy. Therefore, we have between mouse and man, with humans been investigating the cooperation of Apc and cancers that carry β-catenin activating Kras mutations in models of colorectal cancer. mutations all having low levels of E-cadherin. The additional mutation of Kras exacerbates

40 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE COLORECTAL CANCER AND WNT SIGNALLING 41 DROSOPHILA APPROACHES TO CANCER www.beatson.gla.ac.uk/marcos_vidal

We utilise the fruit fly Drosophila to understand fundamental Figure 1 myofibrils and, specifically in the case of animals Scribble mutants are bloated and with tumours carrying both a mutation in enlarged (top), their fat body aspects of cancer biology. We have previously reported an scribble and activation of the Ras oncoprotein, appears more translucent inflammatory reaction from the innate immune system to the (middle panels) and they are we also observed intramyocellular more buoyant than control accumulation of lipid droplets in the muscle (Fig. rise of tumours, comparable to the one observed in cancer genotypes (bottom). Eiger 2). This latter is a remarkable characteristic of mutation suppressed these muscle cachexia described recently. The patients. Importantly, powerful fly genetic tools allowed us phenotypes. transcriptomic profile of the larval cuticle, to define for the first time the contexts dictating ‘good’ versus enriched for the body wall muscle, indicates the ‘bad’ inflammation. Recently, we have dissected a systemic activation of several immune pathways. Our tractable Drosophila model will allow Group Leader crosstalk between tumours and peripheral energy stores Marcos Vidal manipulations of these pathways in the muscle such as the adipose tissue. Remarkably, our data demonstrate of tumour bearing animals to probe for effects Research Scientists both in muscle degeneration and tumour size. Federica Parisi that adipocytes can sense tumours at a distance and react by Alessandro Scopelitti activating proto-inflammatory pathways that act as tissue Publications listed on page 86 Scientific Officer non-autonomous tumour suppressor factors. We have Yachuan Yu characterised many aspects of cancer cachexia in our model; Graduate Students Christin Bauer these results could shed light on the complex systemic Joseph Hodgson relationships between tumours, chronic inflammation and peripheral tissues in human cancer patients.

Drosophila model of cancer cachexia animals, as a proxy for lipid content, was altered Cancer-related cachexia, also known as energy in the mutant animals (Fig. 1). Most importantly, wasting, is perhaps best defined as a ‘loss of the fat body phenotypes observed in tumour body weight that cannot be reversed bearing scribble mutant animals were nutritionally’. Nevertheless, whether this clinical significantly rescued in double mutants for wasting syndrome is a passive bystander effect scribble and eiger/dTNF, the Drosophila in cancer, or whether it plays a positive or homologue of tumour necrosis factor (TNF) negative effect on cancer progression, is (Fig. 1). These results indicate that TNF has an Figure 2 currently unknown. We have characterised key ancestral and evolutionary conserved role in Skeletal muscle from tumour aspects of cancer cachexia in tumour bearing inducing cachexia. This model allows bearing larvae displays increased Drosophila. These include the mobilisation of manipulation of lipid metabolism in adipocytes accumulation of lipid droplets. Note the puncta of Red Oil triglycerides stored in lipid droplets in the fat to examine not only tissue autonomous effects staining in the experimental body, followed by an increase in free fatty acids in the adipose tissue but also in the epithelial genotype (right). and diacylglycerol in the circulation. A tumours. transcriptomic profiling of the fat body, a tissue akin the vertebrate liver and adipose tissue, Effects in the skeletal muscle demonstrated the upregulation of several In cachexia, a loss of mass of lean tissue occurs enzymes involved in lipid catabolism. Most alongside the loss of adipose tissue. Indeed, importantly, the adipocytes, the cell type that skeletal muscle displays a severe degeneration constitutes the fat body, displayed an aberrant in cachectic patients. Our data suggest that the morphology and became translucent, body wall muscle of Drosophila larvae, which is consistent with the biochemical and genetic analogous to human skeletal muscle, also data and pointing towards an evolutionary reacts to high tumour burden and degenerates conserved wasting upon the development of progressively. We observed induction of tumours. Moreover, the buoyancy of the autophagy, reduced density of F-actin in

42 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE DROSOPHILA APPROACHES TO CANCER 43 VASCULAR PROTEOMICS www.beatson.gla.ac.uk/sara_zanivan

Angiogenesis is the process of vessel growth from pre-existing ones and an established hallmark of cancer. Many tumours are indeed highly vascularised. When tumour vessels are intact and functional, they provide the oxygen and nutrients necessary to sustain the uncontrolled proliferation of tumour cells as well as a way for tumour cells to escape the primary tumour and form distant metastases. Conversely, when the Group Leader tumour vasculature is aberrant, vessels are leaky and poorly Sara Zanivan functional and this results in tumour hypoxia, reduced Figure 1 this is required to maintain physiological specifically expressed or with altered levels in Research Scientists Fatty acid oxidation (FAO) levels of endothelial and vascular permeability. the tumour stroma. In particular, we have efficiency of chemotherapeutic drug delivery and limited controls endothelial and vascular Francesca Patella Importantly, vascular hyperpermeability is optimised protocols to perform in-depth Juan Ramon Hernandez permeability. (a) Schematic efficacy of radiotherapy. overview of the formation of a typical of diseases such as cancer, thrombosis quantitative proteomic analysis of soluble Scientific Officer new blood vessel from a and atherosclerosis. Therefore, we have proteins and ECM components secreted by Lisa Neilson pre-existing one induced by identified FAO as a promising metabolic fibroblasts in culture. This approach led us to For these reasons considerable effort has been regulating angiogenesis and hint at strategies pro-angiogenic factors. (b) pathway for further investigation as a target identify more than a thousand proteins secreted Schematic overview of the Graduate Students devoted to understanding the angiogenic to therapeutically interfere with it. to control vascular permeability in by these cells. Among these, we detected ECM Fernanda Kugeratski process in cancer in order to develop therapies approach used to predict metabolic fluxes regulated pathological conditions. components and growth factors, such as Steven Reid that interfere with this process and could be MS-based proteomics has the potential to during the formation of a collagen, fibronectin and transforming growth used in conjunction with conventional chemo- contribute to a better understanding of cell vascular-like network in vitro. Unravelling novel regulators of tumour factor beta that are well known CAF markers and and radiotherapy. Our group exploits its metabolism (Reid et al., Methods Enzymol 2014; The approach integrates stiffness (in collaboration with Jim which were highly abundant in iCAFs compared quantitative mass spectrometry experience with high resolution mass 543: 235) and we have applied this technology Norman’s group) to iNFs. Unexpectedly, we identified a member spectrometry (MS) and accurate quantification in the context of angiogenesis. We have (MS)-based proteomics data with genome-wide metabolic Tumours are composed of many different cell of the chloride intracellular channel protein methods, including SILAC (stable isotope previously used an in vitro model of modelling. (c) The model types in addition to tumour cells. These cells, family to be an ECM component secreted by labelling with amino acids in cell culture), in angiogenesis, which recapitulates aspects of predicts that when the vascular- together with the extracellular matrix (ECM), CAFs. Detailed analysis of this protein revealed combination with a variety of cellular and vessel growth in vivo, where ECs assemble into a like network is fully formed, form what is called the tumour stroma. Among that it is able to enhance angiogenesis and endothelial cells decrease their molecular approaches to determine the vascular-like network within a day, and these stromal cells, we are particularly interested tumour cell invasion and that this process is complex signalling driving (tumour) vessel measured time-resolved proteomic changes glycolysis (blue, dashed line), while increasing their FAO (red, in cancer-associated fibroblasts (CAFs). CAFs mediated, at least in part, by its capability to formation, with a particular focus on endothelial that occur in these cells during this process thick line). (d) Hyperpermeability can secrete a plethora of factors, including ECM regulate the stiffness of the ECM. While most of cell metabolism and the impact of tumour (Zanivan et al., Mol Cell Proteomics 2013; 12: (measured by Evans blue components and ECM modifiers that alter the the current studies have been performed in stroma surrounding newly forming vessels on 3599). We observed that, when cells were extravasation) observed upon mechanical properties of a tumour. Notably, vitro, we are now exploring the relevance of acute inhibition of FAO in this process. assembled into a fully formed network, the high stiffness enhances angiogenesis and these findingsin vivo. This will hopefully reveal a levels of the vast majority of metabolic proteins established blood vessels of the mouse ear. tumour cell invasion, and has a significant novel mechanism through which CAFs regulate Fatty acid oxidation regulates changed. This suggested that major metabolic impact on tumour development. Therefore, it is tumour stiffness. vascular permeability alterations were occurring during network important to better understand the molecular Endothelial cells (ECs) line the inner layer of the formation. In collaboration with Eyal Gottlieb’s mechanisms regulating tumour stiffness. Our Publications listed on page 87 blood vessel wall and are thus in direct contact group and Eytan Ruppin’s group (University of lab uses unbiased proteomic approaches in with the blood (Fig. 1). This location means that Tel Aviv), we used our quantitative proteomic combination with functional in vitro and in vivo ECs are exposed to the changes in oxygen and data to build up a predictive metabolic model of assays to identify and characterise previously nutrient levels that occur in both physiological the cells forming capillary-like structures. We unknown mechanisms through which CAFs and, importantly, pathological conditions such showed that when they are assembled into a regulate the stiffness of the ECM. as cancer. Notably, oxygen and nutrients fuel fully formed network, ECs increase their fatty cell metabolism, and it is therefore important to acid oxidation (FAO), while decreasing their We are exploiting established cell lines of understand if and how cell metabolism affects glycolysis. Moreover, we observed that inhibiting human mammary fibroblasts of different EC function. Currently, the details of how CPT1A, the rate-limiting enzyme for FAO, origins, normal (iNF) and cancer associated metabolism controls EC function are still largely disrupted the network. Using in vitro and in vivo (iCAF), to provide a detailed picture of proteins unknown but a better understanding of this functional studies we determined that ECs use should unravel novel molecular mechanisms FAO to fuel the tricarboxylic acid cycle and that

44 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE VASCULAR PROTEOMICS 45 DRUG DISCOVERY

CANCER RESEARCH UK BEATSON INSTITUTE

Martin Drysdale - Drug Discovery Programme

46 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE DRUG DISCOVERY PROGRAMME 47

Site 1

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Figure 2 DRUG DISCOVERY PROGRAMME Optimised robust bundling assay BDP (µM) 42000

demonstrating concentration 0 25 50 100 200 0 38000 www.beatson.gla.ac.uk/ddp response of a BDP fascin + + + + + + + + + + - - Fascin (A.U)

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absence of inhibitor, >90% of Fascin 26000

50 fluorescence actin is in the pelleted fraction 22000 (P), with little in the supernatant Actin 37 Alexa (S). This is reversed upon addition 18000 of the inhibitor. P S P S P S P S P S P S 14000 % Bundling 90.3 89.2 74.7 19.3 8.2 4.4 0 60 120 180 Figure 3 % Inhibition 0 1.3 17. 78.6 90.9 na BDP-10300 BDP (µ(uM)M) 3 Bead-based fascin actin FigureFigure 2. 2 Figure 3 Drug Discovery sits at the interface between bench science bundling assay. His tagged fascin Figure 3. is pulled down with nickel coated RAS RAF and RALGDS to pathways that control beads and bound actin detected Approximately a third of all human cancers and cellular growth, apoptosis, survival and and clinical development. Our remit is to provide a mechanism with a fluorescent phalloidin. In by which we can translate the fundamental biology being the presence of the BDP fascin the42000 majority of pancreatic, colorectal and lung differentiation. inhibitor compound, bundling by cancers 38000 are driven by mutations in RAS genes.

(A.U) undertaken within the Beatson into new medicines for patients fascin is inhibited and the The34000 RAS gene family has three main members; Multiple strategies have been employed to amount of phalloidin-labelled KRAS,30000 HRAS and NRAS, and all play a pivotal role develop inhibitors that block RAS signalling where there is a clear unmet medical need. actin reduced. in 26000cell signalling. Under circumstances where either directly or through downstream signalling

fluorescence Figure 4 RAS22000 genes are mutated, cells multiply mechanisms. However, none have proved

Alexa Exploiting the basic research themes of the prognostic marker for multiple types of cancer Evolution of a weak binding uncontrollably18000 and escape cell death signals. clinically effective to date and KRAS mutant fragment hit (NMR Kd >1000mM) Group Leader Institute’s scientists we have made significant but is also a compelling drug target. The14000 significance of KRAS mutation in driving cancers remain among the most refractory to to more potent analogues (Kd 41 and maintaining0 60 oncogenesis120 is 180well available treatments. We aim to block the advances in our portfolio targeting novel & 30mM); small molecules that BDP-10300 (uM) Martin recognised. KRAS mutations BDP (µM) are associated with interaction between KRAS and its effector approaches to modulate disease-relevant Fascin is a uniquely folded actin bundling directly bind to KRAS (protein over 20% of all human cancers, and in particular proteins to inhibit oncogenic KRAS signalling. Drysdale processes. protein whose regulation by PKC is tightly in grey). Figure 3. coupled with integrins and the extracellular are associated with the vast majority of Currently, our KRAS programme is in early

Chemistry Metastasis is responsible for approximately 90% matrix. It exists in equilibrium between the Figure 5 pancreatic ductal adenocarcinomas and a hits-to-leads and the team is progressing Catherine Barber The GDP-GTP catalytic cycle; significant number of other tumour types towards more potent small molecule inhibitors Simone Belshaw of cancer deaths, however there are currently cytoplasm and cytoskeleton where it is bound b) The KRAS-GDP:SOS1 including colon and lung. In the GTP-bound with which we believe will be able to Justin Bower no therapeutic agents available to combat this to actin. It has at least two binding sites for nucleotide exchange assay; c) Keneth Davies process. One of the proteins we have chosen to filamentous actin and crosslinks these filaments Two compounds that inhibit and active state KRAS signals from the plasma modulate the function of KRAS. Stuart Francis target is fascin, an actin bundling protein that into tightly packed parallel bundles, oriented nucleotide exchange in the membrane through a functionally diverse set of Duncan McArthur plays a key role in the movement of cancer cells with their growing ends toward the plasma assay represented in 5b. downstream effector proteins including PI3K, Our approach to identifying start points from Angus McMillan which to build a drug discovery project Charles Parry and whose upregulation is known to correlate membrane. Since fascin is found in actin-rich capitalised on our strengths in fragment Angelo Pugliese with poorer overall survival in severe cases of protrusive membrane structures (microspikes Mairi Sime pancreatic ductal adenocarcinoma. We have and filopodia) and degradative structures screening and structure-based drug design. We continued to utilise our fragment-based hit (invadopodia and podosome), which are all performed a primary screen of our fragment Biology identification expertise to target other protein- pro-migratory, it is proposed that by stabilising library against a mutant variant of KRAS using Maeve Clarke NMR KD µ >1000 M NMR KD 41µM NMR KD 30µM high field NMR experiments, identifying a Diane Crighton protein interactions since these are very actin, fascin provides cells with invasive number of weak binders. After confirmation of Dan Croft challenging biological targets but with a high properties that may confer increased Figure 4 . binding, crystal structures were obtained that Patricia McConnell degree of validation as cancer targets. In metastatic potential. NMR KD µ >1000 M NMR KD 41µM NMR KD 30µM Heather McKinnon particular we are targeting RAS, one of the most have been instrumental in enabling us to Mokdad Mezna Figure 4 highly validated cancer targets that is mutated We have taken a fragment based approach to Figure 4 . improve potency (Fig. 4) and a strategy to P increase this further, towards molecules that will Structural Biology in over 30% of all human cancers. identify novel binders of fascin; coupled with a GAP GTP hydrolysis be able to probe inhibition of KRAS effector Kenneth Cameron highly successful crystallography campaign this a) Gillian Goodwin Fascin has enabled us to progress initial fragment hits P protein interactions at the cellular level. Chris Gray GAP Fascin is a migration promoting protein that is through to compounds with low µM binding. To a) RAS GTP hydrolysis RAS RBD Effector Marta Klejnot frequently upregulated when epithelial tissues date, over 100 new small molecule/fascin GDP GTP In order to assess the ability of our compounds Jen Konczal become malignant. 80-90% of cancers are of crystal structures have been generated that to inhibit one of the functions of KRAS, we have Alexander Schuettelkopf RAS RAS RBD Effector GEF GTP binding assayed multiple examples in a nucleotide epithelial origin and fascin is overexpressed in a have aided hit validation and helped to guide GDP GTP exchange assay (Fig.5). variety of tumour types including bladder, structure-based hit-to-lead chemistry (Fig. 1). GDP GTP colon, lung and pancreas. Thus, it is not only a b) GEF GTP binding SOS1 SOS1 Through the application of structural biology Significant progress has been made in the GDP GTP RAS + GDP and medicinal chemistry the project has Figure 1 equally challenging area of biochemical assay b) SOS1 RAS RAS Site 1 GDP GDP SOS1 SOS1 Structure of fascin indicating development. We have developed two new improved potency of the initial fragment hits, RASmantGDP+ GTPγS GTPγS GDP two ligand binding sites (Site 1 screens based on highly validated bundling c) SOS1 RAS RAS delivering compounds that bind directly to KRAS GDP GDP and 2) and their proximity to 100 and inhibit the function of nucleotide exchange. techniques, which has enabled quantitative and 100 functionally important residues 90 mantGDP GTPγS GTPγS c) Future strategies will build on this, utilising a displayed in green and salmon. reproducible assessment of novel compounds 80 80 (%) (%) 70100 Compound A Compound B (Fig. 2 and 3). Combining biochemical data with 100 combination of chemistry, structural biology 60 90 EC50 = 57 uM 60 EC50 = 79 uM 50 80 80 and biology to generate potent KRAS binders crystallography analysis has enabled us to 40 (%) Site 2 (%) 40 70 Compound A Compound B Fluorescene 60 fluorescence 60 that can inhibit the interaction between understand the functional implications of 30 EC50 = 57 uM 20 EC50 = 79 uM 50 mant 20 mant 40 GTP-bound KRAS and its effector proteins. different binding modalities and focus 40 0

Fluorescene 10 fluorescence 30 0 20 mant optimisation to the most promising 20 mant -20 10 1 10 100 0 1 10 100 Compound BDP-00010665-001-002 Concentration (uM)uM / Compound BDP-00010475-001-002 Concentration (uM) uM / Publications listed on page 79 chemical structures. 0 -20 Figure 5 1 10 100 1 10 100 Figure 5 . Compound BDP-00010665-001-002 Concentration (uM)uM / Compound BDP-00010475-001-002 Concentration (uM) uM / Figure 1. 48 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE DRUG DISCOVERY PROGRAMME 49 Figure 5 .

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ADVANCED TECHNOLOGIES

CANCER RESEARCH UK BEATSON INSTITUTE

Kurt Anderson - Beatson Advanced Imaging Resource (BAIR) Gabriela Kalna - Bioinformatics and Computational Biology Gillian Mackay - Metabolomics Gaurav Malviya - Nuclear Imaging Sergio Lilla and David Sumpton - Proteomics and Mass Spectrometry Emma Shanks - RNAi Screening Karen Blyth - Transgenic Models of Cancer Douglas Strathdee - Transgenic Technology

ADVANCED TECHNOLOGIES 51 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 Bioinformatics unit provides support for a range of and cancer research. The development of genetically research projects that require computational methods, encoded fluorophores has revolutionised research by advanced statistical analyses and mathematical modelling. enabling direct visualisation of any gene product. There have Despite the continuing demand for analysing are exploring new analytical approaches and a also been tremendous advances in fluorescence imaging data from high throughput technologies, we wide range of open source pipelines with an technology to visualise molecular dynamics in living cells, strive to ensure that even the smallest task aim to achieve faster alignment and more tissues and organisms. receives our full attention in terms of extensive downstream data analysis. A Head Head experimental design, the application of webserver for viewing sequenced data through appropriate statistical tests and the clear Integrative Genomics Viewer and UCSC Kurt Anderson These powerful techniques are increasingly Imaging’ and start of the BBSRC funded one Gabriela Kalna presentation of results for use in theses Genome browser has been also set up, allowing and publications. our scientists to inspect coverage, aligned reads Scientific Officers sought by researchers, who require assistance in ‘Super-resolved, three-dimensional, multi- Informaticians Tom Gilbey both the evaluation and application of imaging fluorophore tracking of live cell dynamics’. The Ann Hedley and junction counts for their samples. Ewan McGhee technology in order to address fundamental TSB project helped us to develop a close Matthew Neilson Our team focuses on exploratory data analysis, Margaret O’Prey questions in cancer biology. Our mission is to working relationship with M-squared Lasers and with an ultimate goal of providing insights that Our computational model for pseudopod- David Strachan support basic imaging and the development of introduced us to new technology for enhance our understanding of cancer biology. centred cell migration and chemotaxis is now advanced applications. Basic imaging support multiphoton excitation in the form of We offer routine processing of data, differential capable of simulating phagocytosis, and we primarily consists of training new users in simple semiconductor disk lasers, which have novel expression, supervised and unsupervised have recently been investigating three factors acquisition and analysis techniques, such as wavelength, pulse widths and repetition rates. machine learning, and graph and network that influence a cell’s ability to phagocytose a FACS and immunofluorescence microscopy. We also served as a test site for the new theory-based analyses. Our data analysis and nearby entity: the size of the entity, the Development of advanced applications requires Coherent Discovery Laser, which combines a modelling tasks are performed using the adherence of the entity to the cell and the level close work with users to understand their tuning range of 680 to 1300 nm with a fixed open-source Bioconductor package for R, of attractant that is secreted by the entity. In a scientific questions and help them develop output at 1040 nm. The BBSRC project, in Fortran and MATLAB (most notably the related development, this same computational appropriate imaging strategies. The following collaboration with Alan Greenaway (Heriot-Watt Bioinformatics Toolbox and the Statistics model can now be used simulate a field of cells have been identified through consultation as University), will provide us with a diffraction Toolbox). We make use of analytical routines interacting with one another via contact important at the Beatson: medium throughput grating based optical system that can that have been developed in-house or in inhibition. Our particle-based model for long-term time lapse imaging; high resolution simultaneously focus up to nine planes within a collaboration with our colleagues from the simulating populations of E-cadherin has been live cell imaging; confocal microscopy, specimen into a 3x3 image array on a CCD areas of mathematics, statistics, computer calibrated such that it can faithfully reproduce especially for the use of photo-activation, camera. This will potentially enable rapid science and biology. In addition to Ingenuity the experimentally observed behaviour of each -bleaching and –switching; total internal collection of four-dimensional (X, Y, Z, t) image Pathway Analysis, GeneGo Meta Core and CLC L Cell / E-cadherin mutant. In an effort to reflection fluorescence (TIRF) microscopy; volumes, as well as offering the potential for Genomics Workbench, which are available to all simulate wild type E-cadherin in a consistent intravital microscopy; and fluorescence lifetime axial super-resolution of single fluorophores. researchers at the Beatson, we use the fashion, we are using our current model to imaging for the determination of fluorescence Our plan is to incorporate this multifocal system Oncomine Research Premium Edition database investigate cooperativity between each of the resonance energy transfer (FLIM-FRET). More into a Warwick Open Source Microscope and Oncomine Gene Browser to satisfy the permitted interaction types. recently PET, SPECT and CT imaging were (WOSM) that we are building in collaboration demands of researchers who wish to exploit identified as techniques having great potential to with Nick Carter and Rob Cross (Institute for publicly available datasets. Finally, we have developed and deployed a set support the extensive use of preclinical cancer Mechanochemistry) to perform PALM/STORM of routines that aim to improve the productivity models, and preclinical imaging has now super-resolution microscopy. Over the last year, we have been working on a of researchers in the RNAi Screening facility. Our become an independent core facility headed by number of projects integrating data from routines interface the R software environment Gaurav Malviya (see page 55). Collectively, we Finally, we have continued to ‘shake down’ our multiple platforms, including RNAseq, with the Dotmatics Oracle database, such that provide our users with a powerful technology new LaVision TRIM2 multiphoton microscope. microarrays, proteomics and siRNA screens. researchers can quickly and easily perform toolbox for cellular and molecular level This involved taking delivery of two hybrid There has also been a growing interest in exome calculations on a prescribed set of records and investigations of disease and response to GaAsP PMTs, which can be used for time sequencing and identification of fusion genes. then feed the results back into the database. It is therapy in vitro and in vivo. correlated single photon counting, and an Recently, NextSeq 500 Desktop Sequencer anticipated that this seamless integration will adaptive optics module with software to enable replaced the older Illumina model that had lead to considerable productivity gains within This year, one door closed and another opened integration of multiple mirror shapes into previously been used at the Institute. The new the group. with the completion of the TSB funded project multidimensional acquisition routines. sequencer enables our researchers to perform ‘Optimised Lasers for Multi-colour Multiphoton sequencing at a higher depth. Consequently, we Publications listed on page 81

52 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 53 METABOLOMICS NUCLEAR IMAGING www.beatson.gla.ac.uk/advanced_technologies www.beatson.gla.ac.uk/advanced_technologies

The Metabolomics facility is part of the Cancer Metabolism Nuclear imaging techniques have gained wide acceptance in Research Unit (headed by Eyal Gottlieb) and we work closely recent years for monitoring response to cancer therapy at the with its research scientists. This includes our involvement in molecular level. Nuclear imaging using specific probes allows several external collaborations. We also work with several other non-invasive, longitudinal assessment of biological processes, research groups within the Beatson, providing advice on such as tumour metabolism, cell proliferation, angiogenesis, sample preparation and data analysis, as well as measuring hypoxia and receptor dynamics. metabolite levels in samples. Head Head Nuclear imaging involves the administration of research of novel therapeutics by providing tiny amounts of radioactive tracers followed by proof-of-concept for target modulation. Gillian Mackay We have well-established LC-MS methods analysis. As well as the MS data for the Gaurav Malviya functional imaging using PET (positron emission Nuclear imaging may assist in testing an for targeted metabolomics, analysing metabolites, this software uses adducts, Scientific Officer Scientific Officer tomography) or SPECT (single photon emission underlying hypothesis, inform the rational Niels van den Broek approximately 100 metabolites per sample. We isotopes and fragmentation data to identify Agata Mrowinska tomography). In addition, anatomic information selection of dose and schedule and aid are now establishing a workflow for untargeted unknown metabolites. is often obtained by CT (computed decision-making, and may explain or predict analysis to identify more metabolites that differ tomography) scanning to localise radioactive therapy outcomes. In this area, we have between sample groups. We are also developing methods for GC-MS and tracer uptake within the body. The Nuclear collaborated with Anthony Chalmers and GC-MS/MS analysis using our Agilent triple quad Imaging facility provides PET, SPECT and CT Andrew Sutherland (University of Glasgow), who Using a targeted metabolomics approach, we mass spectrometer. We have methods in place imaging support to a range of in vivo research have developed a novel translocator protein analyse a range of samples types including cell for fatty acids and amino acids. The method of projects. In 2014, we increased our imaging (TSPO) imaging tracer, 18F-AB5186 that is an extracts, medium, plasma, urine, tumour and sample preparation is more complex than for armamentarium beyond 18F-FDG and important target for imaging focal other tissues, and CSF. This year, we have LC-MS, as samples need to be derivitised to performed PET/CT and SPECT/CT using a neuroinflammation in diseases such as brain reduced the analysis time per sample and allow them to be volatile in the GC. The aim is to wide variety of radiolabelled probes, including cancer, stroke and neurodegeneration. We have introduced Thermo’s data analysis software, use this as another system for metabolite 18F-FLT (for cellular proliferation), 18F-NaF evaluated the tumour imaging potential of this TraceFinder. This enables much faster and more determination, being able to measure some and 99mTc-MDP (for bone lesions). Through tracer in a glioblastoma mouse model. flexible data analysis than our previous LCquan different metabolites and distinguish between collaboration with Sally Pimlott and Gerry Additionally, with the same groups, we are software. We use our own compound compounds of the same mass that co-elute on Gillen (NHS Greater Glasgow and Clyde), David evaluating another tracer, 123I- FZ044 for SPECT databases of over 100 metabolites in routine the LC systems, to gain a more complete O’Hagan (University of St Andrews), Eyal imaging of poly (ADP-ribose) polymerase LC-MS analyses, identifying known metabolites coverage of the human metabolome. Gottlieb and Kurt Anderson, we have begun (PARP). This tracer is particularly important by their accurate mass and their retention time PET imaging lipid synthesis using 18F-acetate because PARP inhibitors are being investigated on the LC column. Often a comparison of Currently, we have two Thermo Scientific in a colon cancer model. 18F-acetate, an in clinical trials as a means of sensitising tumours metabolite levels between samples is sufficient, LC-MS systems with Exactive mass analogue of acetate, is metabolised to to chemotherapy. however we can quantify the concentrations of spectrometers and a Thermo Scientific fluoroacetyl-CoA and then fluorocitrate, known metabolites in samples using Q-Exactive LC-MS/MS system. These are which cannot be further metabolised to CO In addition, we are at the final stages of commercial standards. For metabolic flux Orbitrap mass spectrometers, which have high 2 and water and is, hence, trapped in the cell in negotiation with two commercial companies to analysis, the fate of heavy isotope tracers (often resolution and can determine extremely proportion to oxidative metabolism. obtain novel radiolabelled probes for nuclear 13C labelled glucose added to medium as a accurate masses. We are in the process of imaging. We have recently published a research nutrient) can be determined by measuring purchasing another Q-Exactive instrument to Recent studies have demonstrated that well article with Jennifer Morton (Morran et al., Gut isotopologues of many intracellular metabolites. replace one of the Exactives. These are validated radiotracers may help in early stage 2014; 63: 1481), where 18F-FLT imaging in KC complemented with our Agilent GC-MS/MS PTEN mice demonstrated how proliferative We are starting to become more involved in triple quad instrument. Figure 1 arrest following rapamycin treatment could be untargeted analysis for cell culture samples and Representative 18FLT PET/CT monitored using 18F-FLT PET scans (Fig. 1). In also extending this to analysing clinical samples, To technically share LC-MS knowledge, the images show pancreatic tumour the future, we look forward to using further to investigate the clinical relevance of in vitro metabolomics team (which includes a member uptake (white arrows) as well as excreted tracer in the bladder novel radiotracers to image the tumour results. Our new Q-Exactive LC-MS/MS of both Eyal Gottlieb’s group and Karen (arrowheads) in a KC PTEN microenvironment, angiogenesis, hypoxia instrument, installed in January, is proving very Vousden’s) regularly meet with Jurre mouse at time of presentation and apoptosis. useful, with its fragmentation capability, in Kamphorst’s lipidomics group and the (left panel) and after 4 days particular to help with the identification and proteomics teams at the Beatson (David of rapamycin treatment Publications listed on page 83 structure of unknown metabolites. We have very Sumpton, Sergio Lilla and Sara Zanivan). (right panel). recently purchased Nonlinear Dynamics’ Progenesis software for untargeted data Publications listed on page 83

54 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 55 PROTEOMICS AND MASS SPECTROMETRY RNAi SCREENING www.beatson.gla.ac.uk/advanced_technologies www.beatson.gla.ac.uk/advanced_technologies

The proteomics facility has provided expertise in the The RNAi Screening facility couples high throughput RNA characterisation of proteins by mass spectrometry since its interference (RNAi) screening and drug repurposing launch in 2005. Our key goal is to facilitate proteomic analysis approaches with high content imaging (HCI) to translate within the Institute by offering access to state-of-the-art fundamental cancer research towards new therapies. instrumentation with assistance in sample preparation, analysis and interpretation of experimental data. Collectively, these approaches provide a In early 2014, our lab relocated to the Wolfson powerful tool for identifying novel or key Wohl Cancer Research Centre, where we have players in a system of interest and elucidating additional space to incorporate our expanding Sergio Lilla The facility is well equipped with two mass We are continually striving to improve the Head novel drug targets and/or drug partners to suite of instrumentation and a more substantial spectrometers, an Orbitrap Velos system crucial methods currently in place to enrich the Emma Shanks improve existing cancer therapeutic dedicated tissue culture resource. We also coupled to an Easy-LC nano HPLC and an quality of the results the facility can provide. The approaches. received funding for a second HCI instrument, AB-Sciex 5600 triple TOF system with Eskigent recent introduction of parallel reaction Scientific Officers which has provided much needed extra UPLC. The facility also houses a number of monitoring (pRM) encapsulates this concept. Kay Hewit1 Our activities include development of high resource to support our throughput but has also Lynn McGarry dedicated software platforms. These include: a The pRM approach provides at least two orders throughput RNAi screening (HTS) assays, which brought us additional confocality capability for Mascot server for protein identification; a of magnitude more sensitivity than conventional involves scaling up existing cell-based assays to use within screens and in supporting hit dedicated MaxQuant server for the analysis of LC-MS. This boost in sensitivity allows us to Informatics Manager Daniel James produce robust, appropriately controlled validation and deconvolution. SILAC data; Progenesis and Skyline for the identify and quantify protein and their screening assays, and refining the use of analysis of label-free, SWATH or pRM data; and modifications within samples that otherwise Graduate Student additional outputs to provide multiparametric Through collaboration, we have supported Scaffold for data compilation and dissemination. would be lost. Another recent improvement, Matthew Davidson These instruments and software packages the use of high pH reverse phase LC HCI analysis. We run HTS assays using robotics development, purchasing and implementation together allow the facility to provide a broad fractionation, has allowed us to delve deeper 1 CRUK Glasgow Centre and automation to bring precision, accuracy of a large metabolism-focused siRNA library as a David Sumpton range of proteomic workflows, from simple into the proteome, identifying more proteins and throughput to the process. Data analysis is Centre-wide resource, as well as some smaller single protein identification to complex than was previously possible in our hands. an important part of large-scale HTS work; we bespoke sets. These new sets have already been proteome-wide protein identifications. have data management solutions and databases widely utilised. We continue to work to Research Scientist More targeted experiments such as the Publications listed on page 82 in place to support this, and carefully evaluate implement prebuilt and develop bespoke Emma Carrick characterisation of both post-translational and deconvolute data to give quality assurance. algorithms to allow calculation of additional and chemical protein modifications can be parameters from screening assays. Notably this Scientific Officers undertaken. With the incorporation of SILAC Sergio Lilla During 2014, we ran 13 screening campaigns in includes cell/nuclear shape, size, area, intensity or with a well-designed label-free experiment collaboration with eight groups across the and proximity, as these phenotypes can be David Sumpton relative protein quantitation can also be CRUK Glasgow Centre. These encompassed indicative of additional mechanistic processes. performed across all workflows. The facility both large-scale projects to support target We are also developing algorithms to quantify stores all informatics data (raw data, login forms, etc.) submitted to the facility since 2006, making identification, and more tailored, focused organoids within screening platforms. In it available to Beatson users for consultation, approaches to provide validation to support September, we welcomed Kay Hewit to the method development or publication. other observations. We have addressed the group, as a screening scientist. following questions in multiple cancer backgrounds of both mouse and human origin: We are currently working to advance the scope of phenotypic analysis we can offer. • Genes exerting selective lethality under Further to this, we intend to implement more hypoxic conditions versus normoxic detailed morphological analysis pertaining to conditions re-distribution and re-organisation of cellular • Mediators and effectors of oncogenic Myc shape in response to siRNA knockdown/drug • Potential combinatorial partners for cell lines treatment. Such phenotypic changes have been demonstrating resistance to an innovative demonstrated in response to siRNA treatment in therapeutic agent a cancer background (Yin et al., Nat Cell Biol • Selective mediators of response to different 2013; 15: 860), and implementation of this p53 genomic backgrounds within the facility will be of great utility. • Mediators of chemotherapeutic resistance in Her2+ breast cancer Publications listed on page 86 • Drug repurposing approaches in pancreatic, glioblastoma and oral-derived cell lines

56 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 57 TRANSGENIC MODELS OF CANCER TRANSGENIC TECHNOLOGY www.beatson.gla.ac.uk/advanced_technologies www.beatson.gla.ac.uk/advanced_technologies

Our lab capitalises on the use of in vivo models to investigate The Transgenic Technology Laboratory employs molecular how deregulation of signalling and metabolic pathways genetic approaches to analyse the role of genetic alterations to contributes to cancer development, using these tools to cancer. The use of this technology allows the generation of explore new therapeutic targets which could benefit cancer more precise models of human cancer. patients. The lab has a particular interest in the role of RUNX genes in breast and prostate cancer. Using gene targeting and genome editing, it is expand the range of different techniques we possible to make controlled alterations in employ to refine existing models and generate specific genes in the germline. Combining a new ones. We are continuing to develop shRNA Head Head Preclinical murine models are fundamental leukaemia, is commonly expressed in breast variety of these alterations commonly observed technology to employ this directly in models. Karen Blyth tools for the researchers at the Beatson where cancer and that its expression correlates with Douglas in human cancer allows the generation of more This will allow control of gene expression biological questions can be interrogated in the poor prognosis in the triple-negative subtype sophisticated and accurate cancer models. directly in tumours in situ, allowing, for example, Research Scientists context of a physiologically relevant system. (Ferrari et al., PLoS One 2014; 9: e100759). While Strathdee potential therapeutic targets to be effectively Kirsteen Campbell1 With genetic models we can accurately recreate these studies point to a pro-oncogenic role for The use of stem cells to generate validated in tumour models. Nicholas Rooney Scientific Officers tissue specific changes to mimic the alterations RUNX1, this gene may also have tumour Laurence Cadalbert cancer models Farah Naz Ghaffar Scientific Officers found in common cancers such as colorectal constraining properties and we are currently Embryonic stem (ES) cells provide a very useful While shRNA technology may increase the Dimitris Athineos and breast cancer to better understand these exploring the paradox of this behaviour and its Graduate Student tool for the analysis of gene function in cancer. range of model types we can generate the rise Susan Mason diseases. In addition, we have good models for relevance in breast cancer. We have found that Nicola Laprano Firstly, these cells have relatively high rates of of genome editing technology (CRISPRs and melanoma and pancreatic cancer, disease types the related gene, RUNX2 is also expressed in DNA recombination and this allows us to make TALENs) has the potential to significantly Clinical Research Fellow Anne McKillop2 currently associated with poor outcome and breast cancer, albeit in a more restricted controlled alterations in specific genes, which increase the efficiency with which models can limited treatment options; so we can use our subgroup of triple-negative patients, but is mimic those detected in human cancers. be generated (Fig. 1). This may not only impact Graduate Students models to investigate associated signalling associated with reduced cancer-specific survival Secondly, ES cells can be differentiated into a the speed with which we can generate models Nicola Ferrari pathways and novel therapies. A distinct (McDonald et al., Dis Model Mech 2014; 7: 525). wide variety of different tissue types, which but also has the potential to allow the creation Alessandra Riggio advantage to these in vivo models is their The WNT/β-catenin pathway, an important allows the consequences of any genetic of multiple genetic alterations simultaneously. If Intern representative tumour pathology and an intact signalling node in epithelial cancer, is also alteration to be tested not only in the stem cells this technique proves to be as powerful as Hannah Lawson3 tumour microenvironment that can be lacking associated with triple-negative breast cancer but also in the specific tissues in which the suggested by initial experiments, this could have in tissue culture systems but which affects how and RUNX2 is strongly expressed in WNT-driven cancer arises. We are currently collaborating on a substantial impact on how models are 1 Royal Society Dorothy tumours develop and respond to treatment. The models of breast cancer. RUNX2 contributes to a number of projects using strategies such as generated and how cohorts are produced for Hodgkin Fellowship lab leads the way in exploiting these resources the regenerative potential of the mammary point mutations or conditional knockouts to ongoing experiments. We are currently trying to 2 CRUK Glasgow Centre (joint with School of and collaborates with the Beatson groups to stem cell, where interestingly WNT signalling is uncover the roles of a variety of genes in the implement this technology effectively and also Veterinary Medicine) dissect all stages of disease progression. In also important, and demonstrates a progression of different cancers. adapt aspects of it to make it more directly 3 from University of Glasgow particular, we have been working closely on commonality between these pathways in the applicable to developing the types of tumour several projects with Karen Vousden and Eyal regulation of mammary tissue. We are also Adopting a flexible approach to models most frequently used here. Gottlieb on strategies to better understand interested in the role of MCL1 in breast cancer Figure 1 improving efficiency cancer cell metabolism and its relevance in vivo, and were awarded Breast Cancer Campaign CRISPR-mediated genetic As well as making the most of current This year, in order to improve efficiency, alteration in mouse embryonic such as assessing how loss of the TCA cycle funding this year to explore this further. technology, we are also trying to improve and welfare and distribution of models, we have stem cells. (A) Diagram of mouse enzyme fumarate hydratase predisposes to Taz genes and design of CRISPR been establishing methods for cryopreservation renal cell carcinoma (Zheng et al., Nat Commun Publications listed on page 78 guide oligos to exon 3. Red and re-derivation. Now up and running, both 2015; 6: 6001) and how serine depletion may be arrows indicate the target sites. methods will facilitate the exchange of a novel therapeutic approach (Maddocks et al., (B) Screening ES cell clones by reagents between the Beatson and other labs. Surveyor nuclease assay Nature 2013; 493: 542). In the same vein, we are also trying to make detected mismatches in the modified alleles. (C) the most use of resources available from a Defining the role of the RUNX genes in Confirmation of Surveyor results variety of external sources. Where possible, breast cancer using restriction enzyme analysis we are continuing to use cell lines from the Members of the lab have been specifically of PCR product. From each IMPC and are now importing GEMM-ESCs clone, control (C) and SfaN1 interested in elucidating the role of RUNX1 and from the Netherlands Cancer Institute via digested (S) PCR products are RUNX2 in epithelial cancer. We have shown that visualised. (D) Sequence of Infrafrontier. These stem cell lines already RUNX1, renowned for its importance in human mutations recovered in exon 3 of carry a number of genetically modified alleles the Taz gene following CRISPR and we will further modify them to allow them transfection of mouse to be effectively employed here for the analysis embryonic stem cells. of cancer development.

58 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE ADVANCED TECHNOLOGIES 59 BEATSON ASSOCIATES

UNIVERSITY OF GLASGOW

Peter D. Adams - Epigenetics of Cancer and Ageing David Bryant - Molecular Control of Epithelial Polarity Jurre Kamphorst - Cancer Metabolomics Daniel J. Murphy - Oncogene-Induced Vulnerabilities Stephen Tait - Mitochondria and Cell Death

BEATSON ASSOCIATES 61 Figure 2 chromatin-remodelling complex. Our results EPIGENETICS OF CANCER AND AGEING DNA methylation gains and losses map the distribution of the HIRA chaperone in senescent cells overlap with across the chromatin landscape and point to www.beatson.gla.ac.uk/peter_adams hyper- and hypomethylation in cancer. Paradoxically, this different interacting partners at functionally suggests that the epigenome of distinct regulatory sites (Pchelintsev et al., Cell senescent cells might be primed Rep 2013; 3: 1012). for progression to cancer. In turn, this suggests that senescence These studies are being extended to an analysis might be an imperfect tumour suppressor mechanism, and of HIRA, histone H3.3 and other chromatin accumulation of senescent cells features in senescent cells. We have performed with age might predispose to ChIP-seq and DNA methyl-seq of several cancer. The reason(s) why cancer histone modifications and DNA methylation in increases with age in humans is proliferating and senescent cells (e.g. Shah et al., The Adams lab investigates the impact of chromatin structure poorly understood, at present (Cruickshanks et al., Nat Cell Biol Genes Dev 2013; 27: 1787). These studies have and epigenetics on cell proliferation, ageing and cancer. In 2013; 15:1495). revealed remarkable and paradoxical insights particular, we hypothesise that age-associated changes in into chromatin in senescence, and the role of Figure 3 senescence as a tumour suppressor and its The altered epigenome of chromatin structure, function and regulation contribute to the contribution to tissue ageing. Mechanistic senescent cells might promote dramatic age-associated increase in incidence of cancer. While age-associated increase in hypotheses are being tested, and these analyses incidence of human cancers. Our are being extended to studies of human and age is the biggest single risk factor for most cancers, the reason analyses and previous studies and mouse aged and premalignant tissues to define ideas lead to the following model: the mechanism by which age-associated for this is current poorly understood. Senescent cells accumulate with Group Leader chromatin changes predispose to cancer. age in human tissues. These Peter D. Adams senescent cells harbour low-level Cell senescence is an irreversible proliferation chromatin structure, and onset of diseases methylation of CpG islands of Senescent cells appear ‘epigenetically Beatson Associate arrest instigated by a variety of molecular of ageing, including cancer. tumour suppressor genes that is primed’ to form cancer cells triggers including acquisition of activated insufficient to silence gene Research Scientists Altered DNA methylation and associated expression. However, even a John Cole oncogenes, and shortened telomeres caused Genome-wide analysis of chromatin destabilisation of genome integrity and function limited/transient escape from sequencing. To complement this analysis of Dina Dikovskaya by excess rounds of cell division. In addition, structure and chromatin regulators in senescence, for example due to epigenetic marks in senescence, we are also is a hallmark of cancer. Replicative senescence Maria Grazia Vizioli senescent cells secrete a cocktail of senescent cells genetic inactivation of PTEN exploring the genome-wide distribution of is a tumour suppressor process that imposes a Farah Jaber-Hijazi inflammatory cytokines, chemokines and matrix To better understand the structure and function confers additional rounds of cell limit on the proliferative potential of normal cells Francisco Marques histone chaperones in senescent cells, again proteases (the ‘inflammatory secretome’ or of chromatin in senescent cells, we are division that permits proliferation- Mohammad Iqbal Rather using state-of-the-art approaches. We have also that all cancer cells must bypass. Here we show dependent spreading of DNA Neil Robertson senescence-associated secretory phenotype, performing genome-wide analyses of histone by whole-genome single-nucleotide bisulfite methylation. Hence, an initial collected gene expression data to build a SASP) that is capable of influencing behaviour of modifications and DNA methylation to compare ‘seed’ of methylation in a comprehensive, integrated view of the sequencing that replicative senescent human Clinical Research Fellows neighbouring cells, including immune cells. chromatin in proliferating and senescent cells. senescent cell in an aged tissue epigenetic control of senescent cell function. cells exhibit widespread DNA hypomethylation Annie Latif Compelling evidence now indicates that cell To do this, we are using next generation can, in conjunction with other and focal hypermethylation. Hypomethylation Douglas MacKenzie senescence is a potent tumour suppression sequencing (ChIP-seq), microarray and genetic alterations and clonal To initiate studies in this area, we have analysed occurs preferentially at gene-poor, late- selection, grow to full CpG island Scientific Officer mechanism, notably in cells harbouring proteomic approaches and whole genome replicating, lamin-associated domains and is hypermethylation and tumour the HIRA histone chaperone complex. This Claire Brock activated oncogenes. Senescence-associated single nucleotide bisulphite modified DNA suppressor gene silencing, complex, comprised of HIRA, UBN1 and linked to mis-localisation of the maintenance proliferation arrest and the SASP act in concert thereby facilitating progression to CABIN1, collaborates with histone-binding DNA methyltransferase (DNMT1) in cells Graduate Students to achieve tumour suppression: proliferation late-life cancer. Global approaching senescence. Low-level gains of Rachael Hewitt protein ASF1a to incorporate histone variant arrest directly curtails tumour growth and the hypomethylation in senescent Desiree Piscitello1 H3.3 into chromatin in a DNA replication- methylation are enriched in CpG islands, cells may also promote genome SASP calls on innate immune cells to eliminate including at genes whose methylation and instability and dysfunction independent manner. Consistent with this role 1 joint with David Gillespie the offending damaged cells. Because of (Cruickshanks et al., Nat Cell Biol in DNA replication-independent chromatin silencing is thought to promote cancer. Gains senescence, most primary human cells have a 2013; 15:1495). metabolism, we have previously implicated this and losses of methylation in replicative finite proliferative lifespan, and evidence has chaperone in regulation of chromatin in non- senescence are thus qualitatively similar to been presented that senescence contributes to proliferating cells. To better understand HIRA’s those in cancer (Fig. 2), and this ‘reprogrammed’ tissue ageing in vivo, in part by limiting the function and mechanism, we integrated HIRA, methylation landscape is largely retained when proper self-renewal of stem cells and tissues. UBN1, ASF1a and histone H3.3 ChIP-seq and cells bypass senescence. Consequently, the In sum, cell senescence has both beneficial gene expression analyses. Most HIRA-binding DNA methylome of senescent cells might and detrimental effects, and bypass of sites co-localise with UBN1, ASF1a and H3.3 at promote malignancy, if these cells escape the senescence can lead to tumour formation active promoters, and active and weak/poised proliferative barrier. Since senescent cells appear through uncontrolled proliferation of Figure 1 enhancers. At promoters, binding of HIRA/ to be imperfect tumour suppressors, at least damaged cells (Fig. 1). Senescence as a tumour suppressor mechanism. UBN1/ASF1a correlates with the level of gene from an epigenetic perspective, accumulation Acquisition of an activated oncogene or inactivation of a expression. HIRA is required for deposition of of senescent cells in aged tissues might Cellular senescence, ageing and cancer are all tumour suppressor initially causes a proliferative burst. Ultimately, senescence kicks in to arrest proliferation of histone H3.3 at its binding sites. There are contribute to increased incidence of cancer accompanied by marked changes in chromatin the cells harbouring the oncogenic event. Proliferation marked differences in nucleosome and co- with age (Fig. 3) (Cruickshanks et al., Nat Cell structure. We are interested in the epigenetic arrest is reinforced through the senescence-associated regulator composition at different classes of Biol 2013; 15:1495). changes associated with senescence, and their secretory phenotype (SASP). Senescence-associated HIRA-bound regulatory site. Underscoring this, proliferation is likely to arrest tumour progression by contribution to the senescent phenotype. In we report novel physical interactions between Publications listed on page 87 addition, since senescent cells promote ageing, preventing proliferation of neoplastic cells and suppressing accumulation of additional genetic the HIRA complex and transcription factors, a we are testing the hypothesis that senescence- alterations. In addition, senescence recruits the innate chromatin insulator and an ATP-dependent associated changes in chromatin structure immune system to clear the genetically altered cells contribute to age-associated changes in that threaten the host with malignant disease.

62 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE EPIGENETICS OF CANCER AND AGEING 63 MOLECULAR CONTROL OF EPITHELIAL POLARITY www.beatson.gla.ac.uk/david_bryant

Cells of many tissues are polarised, collectively forming 3 - 10 days Single Cell Spheroid with lumen Cyst

configurations tailored to the needs of a tissue. A common Luminal Medium cell feature of most cancers of epithelial origin is the loss of containing ECM Branched Lumen normal tissue architecture. Despite advances in identifying Epithelial Nuclei signalling pathways and genetic alterations involved in Lumen Organ

driving cancer progression, little is known about this very ECM-coated glass basic, yet fundamental question: how are highly organised Group Leader tissues turned into disarrayed tumours? days: 0 2 4 6 8 David Bryant Beatson Associate Our group focuses on how cell polarity is Our group is developing systems for examining Graduate Students controlled in epithelial cells, particularly in gene manipulation in spheroids in a medium- Marisa Nacke1 prostate tumours. Our efforts are focused on to-high throughput fashion, to model genetic Figure 1 Figure 2 1 CRUK Glasgow Centre two molecular pathways: the role of ARF changes seen in patients with metastatic cancer. GTPases (and their regulators and effectors, This will allow us to rapidly determine the which we call the ‘ARF regulome’), and the role genetic changes that facilitate disruption to Figure 1 acquisition of invasive behaviours when We will be using proteomic approaches to By culturing cells on glass- of the cell surface protein, podocalyxin. Both tissue organisation. Our future work aims to oncogenes are expressed. understand the interacting network of proteins bottomed chambers coated with that regulate podocalyxin function, viral genetic molecules are highly overexpressed in develop such systems for prostate, lung, breast extracellular matrix (ECM), we are metastatic prostate cancer tumours. Our lab and kidney spheroids. able to direct single cells to Knowing up from down: control of cell techniques to manipulate its function and focuses on molecular imaging studies to self-assemble into multicellular polarity orientation ultimately mouse models of podocalyxin loss. characterise how these molecules control GTPase regulation of cell polarity spheroid structures with a single, A fundamental feature of an epithelial tissue is These studies are aimed at providing a detailed central lumen. This process normal and aberrant prostate epithelial Polarised epithelial cells are the basic building that the apical surface faces, and forms, the molecular dissection of podocalyxin function occurs over a number (10) days, lumen. We recently uncovered a molecular and its contribution to cancer progression in polarisation. Our ultimate aim is to investigate block of many organs. A typical polarised and allows us to study the these molecules as potential biomarkers of epithelium is composed of a highly organised dynamics of tissue formation. switch that allows cells to sense the orientation vitro and in vivo. prostate cancer in patients and possible targets mono- or bilayer of cells surrounding a single, of where their apical surfaces will be located, i.e. for future therapeutic intervention. central lumen. This organisation requires Figure 2 how to know up from down (Bryant et al., Dev Publications listed on page 88 Three-dimensional cultures of distinct plasma membrane domains, consisting Cell 2014; 31: 171). This pathway involves cells cells to form cysts (also called sensing the ECM, whereby cells repress the Three-dimensional culture models of the apical surface facing the lumen and the spheroids) allow us to model the Three-dimensional spheroid culture, where basal-lateral side contacting neighbouring cells basic structure of epithelial organs localisation of the apical protein, podocalyxin single epithelial cells are grown in extracellular and the underlying ECM. Such organisation is in the laboratory. This allows us to at the ECM interface and redirect it to the proper matrix (ECM)-containing gels to form lumen- often lost as cancer progresses, though the understand and manipulate luminal site. Notably, inhibition of ECM-derived factors known to be altered in surrounding spheroids, can be used to model exact mechanisms as to why this is remain signals causes the development of collective cancer patients and model in the migration and invasion, led by the apical tissue organisation in vitro (Fig. 1). This basic elusive. By using viral genetic tools to laboratory what the effect would polarised unit is reiterated to build the branched manipulate gene expression in spheroids, we be on a tissue’s organisation. surface protein podocalyxin. This represents a epithelial organs that make up many of the have determined that specific membrane completely novel, three-dimensional model of epithelial and endothelial tissues in our bodies trafficking proteins of the Rab GTPase family are collective cell motility. Podocalyxin expression (Fig. 2). Three-dimensional culture has received critically important for the normal polarisation is highly amplified in prostate, breast and much attention in recent years as methods for of epithelial cells and spheroids. In contrast, pancreatic tumours, and is mutated in familial making spheroids out of a variety of sources specific members of the Arf GTPase family are forms of prostate cancer. The mechanisms of have become available, including from cell lines, involved in the disruption of polarity induced by how podocalyxin contributes to cancer stem cells and tumour cells from patients. oncogene expression. Notably, many of these migration and invasion are largely unclear. Our Three-dimensional culture thus allows for genes are altered in metastatic tumours of future work aims to examine the molecular basis formation and growth of ‘mini-organs’ from prostate cancer patients. Our current studies by which podocalyxin controls polarisation of such sources. This presents an exciting new are aimed at investigating how Arf and Rab spheroids, and potentially drives invasion and possibility to examine, in a rapid fashion, the GTPases regulate normal cell polarisation in metastasis in prostate cancer. factors that regulate tissue formation and its spheroids, and how they participate in the disruption in cancer.

64 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE MOLECULAR CONTROL OF EPITHELIAL POLARITY 65 requirements of pancreatic cancer cells, was still CANCER METABOLOMICS normoxia glucose unknown. We therefore performed www.beatson.gla.ac.uk/jurre_kamphorst metabolomic comparisons of human PDAC and benign adjacent tissues, which revealed that lactate pyruvate tumour tissue was low in glucose, upper glycolytic intermediates, creatine phosphate citrate AcCoA Fatty acids and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such α-ketoglutarate accumulation could arise from extracellular proteins being degraded through glutamine macropinocytosis in quantities necessary to Cancer is a disease of uncontrolled cell proliferation. To enable meet glutamine requirements, which in turn would produce an excess of most other amino rapid growth, cancer cells need to alter their metabolism to hypoxia acids. Entirely consistent with this, TMR-dextran glucose generate the required energy and cellular building blocks. acetate (an established marker for macropinocytosis) assays performed on freshly resected human However, while trying to satisfy the metabolic demands of PDAC tissues demonstrated active lactate pyruvate ACSS2 growth, cancer cells often face a limited supply of primary macropinocytosis. When investigating the metabolic consequences of macropinocytosis nutrients and oxygen, creating metabolic stress. Recent studies citrate AcCoA Fatty acids further, we found that cultured murine PDAC show that cancer cells can switch to consuming the abundant cells can grow indefinitely in media lacking Group Leader extracellular proteins and lipids to support growth in these α-ketoglutarate single essential amino acids, when cultured in Jurre the presence of physiological levels of albumin stressful conditions. As these metabolic ‘scavenging’ pathways (Fig. 2). Moreover, PDAC cells were able to Kamphorst glutamine replicate in the total absence of free amino CRUK Career are tumour-specific we hypothesise that they are promising acids. That PDAC cells actually degrade Development Fellow Figure 1 Human pancreatic tumours are nutrient poor extracellular protein to derive amino acids was Beatson Associate candidates for therapeutic intervention. Therefore, our primary Acetate maintains lipogenesis in and tumour cells actively scavenge further demonstrated using isotope-tracing hypoxia. In normoxic conditions aim is to elucidate the enzymes involved in scavenging, and extracellular protein experiments. Growth in amino acid-deficient Research Scientists glucose is the primary carbon Vinay Bulusu their regulation. For this we combine metabolomics, donor for acetyl-CoA production, We recently contributed to the discovery that a medium supplemented with albumin was Sergey Tumanov the substrate for fatty acid cellular process by which cells can internalise characterised by simultaneous glutamine lipidomics and proteomics approaches with sophisticated synthesis. In a low oxygen part of their microenvironment, called depletion and essential amino acid Laboratory Manager (hypoxic) environment, which macropinocytosis, enables cancer cells to accumulation, in accordance with the human Michelle Schug cancer models. typically occurs in tumours, scavenge and degrade extracellular protein to tumour metabolomics results. Hence, protein glucose is instead mainly shunted Graduate Students towards lactate, creating a lack of support their metabolism (Commisso et al., scavenging through macropinocytosis occurs Evdokia Michalopoulou Acetate is a substrate for fatty acid synthesis the α-ketoglutarate to citrate conversion more carbon to maintain fatty acid Nature 2013; 497: 633). Macropinocytosis is in human PDAC and is likely an important supply 1 Nicholas Rabas in hypoxic cancer cells reversible. An alternative explanation of the synthesis and hence growth. By especially prominent in cell culture and in vivo route for amino acids, when primary nutrients performing stable isotope 1 Cell growth requires fatty acids for membrane extensive citrate and fatty acid labelling from models of pancreatic ductal adenocarcinoma and oxygen supplies are low. This makes it an joint with Jim Norman experiments, we showed that synthesis. Fatty acids are assembled from glutamine in hypoxia is isotope exchange (PDAC), a poorly vascularised, KRAS-driven attractive target for therapeutic intervention. acetate contributes substantially two-carbon units in the form of acetyl-CoA without a net contribution of carbon. By feeding to acetyl-CoA, and thus fatty acid, malignancy. However, whether it actually 13 (AcCoA). In nutrient and oxygen-replete hypoxic cancer cells C-tracers and then production in hypoxic conditions. occurred in human pancreatic tumours and to Publications listed on page 88 conditions AcCoA is predominantly derived analysing the fatty acids with mass Acetate assimilation may be an what degree it could support the amino acid from glucose: pyruvate dehydrogenase spectrometry, we were able to identify a attractive target to inhibit the produces AcCoA from glucose-derived substantial contribution (20-50% of the AcCoA growth of hypoxic tumours.

pyruvate in the mitochondria, followed by pool, depending on the cell line) from an Figure 2 ligation of the acetyl group to oxaloacetate to unknown carbon source, i.e. not coming from Pancreatic cancer cells can produce citrate. Citrate is then transported into glucose or glutamine (Kamphorst et al., Cancer proliferate in medium lacking the cytosol and cytosolic AcCoA produced by Metab 2014; 2: 23). Follow up work revealed only amino acids when supplemented ATP citrate lyase. In hypoxic conditions (which a minor contribution of non-glutamine amino with physiological levels of serum protein. (A) Mouse-derived frequently occur in tumours), however, most of acids and of fatty acids to acetyl-CoA in hypoxia. pancreatic cancer cells were the glucose-carbon is shunted toward lactate as Instead, acetate is the major, previously cultured in medium lacking the its entry into the TCA cycle is blocked (Fig. 1). unaccounted for, carbon donor. Thus, acetate essential amino acid leucine and How this affects AcCoA and consequently fatty assimilation is a route by which hypoxic cells additionally with or without acid production in hypoxic cancer cells remains can maintain lipogenesis and thus proliferation. supplementation of additional 5% of bovine serum albumin (BSA). (B) an area of active investigation. It has been Conversion of acetate to AcCoA in the cytosol is Growth curve for the same postulated that reductive carboxylation of catalysed by the enzyme acetyl-CoA synthetase conditions as (A). (C) The same glutamine-derived α-ketoglutarate enables 2 (ACSS2), and complementary work by Eyal cells were cultured in medium hypoxic cells to maintain citrate and AcCoA Gottlieb’s group and others has shown that its lacking all amino acids, with or production. However, it was later noted that inhibition does indeed have potent anti-growth without physiological (5%) BSA. For (B), data are means ± SE, n ≥ 3. dropping citrate levels in hypoxic cells makes effectsin vivo.

66 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE CANCER METABOLOMICS 67 Figure 1 ONCOGENE-INDUCED VULNERABILITIES Induced dependencies need not reflect direct molecular www.beatson.gla.ac.uk/daniel_murphy interactions. Oncogene-induced cell growth, typically requiring signal transduction via the mechanistic target of rapamycin (mTOR) pathway, drives rampant ATP consumption that must be compensated for through increased cellular intake of fuel (e.g. glucose, amino and fatty acids) combined with AMPK- mediated attenuation of macromolecular synthesis. Oncogenic signalling profoundly alters how cells respond to Upon suppression of Ark5, this feedback mechanism is impaired, Myc-induced metabolic vulnerability combined with RNA-SEQ gene expression their environment, typically putting tumour cells under leading to ATP depletion and tremendous pressure to reconcile conflicting cues. For bioenergetic catastrophe. In As part of a coordinated programme of cell analysis to identify a cluster of genes whose principle, any intervention that growth required for cell division, Myc engages a expression increases with progression from example, tumour cells must reorganise their metabolic similarly impairs bioenergetic number of biosynthetic programmes, low- to high-grade lung cancer, many of which homeostasis may selectively kill prominently including ribosome assembly and are frequently amplified and/or overexpressed in pathways to balance competing needs for biosynthetic tumour cells. protein translation, placing tremendous human NSCLC. We are presently combining precursors with energetic homeostasis, commonly while energetic demand upon the cell. In order to functional screening in vitro and in vivo to surviving in a milieu of limiting oxygen and nutrients. Our maintain energetic homeostasis, Myc validate the top 50 progression-associated Group Leader upregulates glucose transporters and glycolytic genes, with pharmacological approaches to overarching hypothesis is that such oncogene-induced enzymes, promoting the Warburg effect of investigate the potential therapeutic impact of Daniel biological perturbations can be exploited for cancer therapy, limited glucose breakdown, and in parallel suppressing specific pathways involved in J.Murphy induces expression of glutamine transporters progression to high-grade disease. This work and exploits this pathway to maintain the citric aims to identify new candidates for targeted Beatson Associate even in the absence of direct suppression of driver oncogenes. We use deregulated Myc as our paradigm oncogene coupled acid cycle. The energetic strain that Myc therapy as well as indicators/biomarkers of early Research Scientist deregulation thus places upon the cell is evident disease progression. Meera Raja with a mixture of candidate and RNAi-based screening in progressive activation of the AMP-activated protein kinase AMPK, which plays a key role in As part of our efforts to further develop lung Scientific Officers approaches to identify induced vulnerabilities in vivo and in maintaining energetic homeostasis. AMPK in cancer research in Glasgow, we have Alan McVie Jacqueline Tait-Mulder vitro and are actively exploring several strategies for selective turn inhibits TORC1 to attenuate the rate of established a Lung Cancer Research Steering elimination of cells that overexpress Myc. macromolecular synthesis, effectively allowing Group in collaboration with Anthony Chalmers Graduate Students cells to balance the rate of ATP consumption and a group of clinician scientists from Tiziana Monteverde with that of ATP production. For reasons that are Gartnavel and the Southern General Hospital, Nathiya Muthalagu presently unclear, an AMPK-related kinase, Ark5 spanning disciplines of Radiation Oncology, Sarah Neidler MYC in cancer suggested that direct regulation of BH3 proteins Jennifer Port Overexpression of the transcription factor Myc presents an alternative pathway to Myc-induced is required for this homeostatic feedback Pulmonary Thoracoscopy and Pathology. The occurs in a huge number of human cancers apoptosis. The stoichiometric balance between mechanism under circumstances of Myc-driven group currently meets four times a year to arising from almost every tissue type. Myc pro-apoptotic and anti-apoptotic BH3 family energetic stress. Depletion of either AMPK or stimulate multidisciplinary discussion, overexpression may arise from focal or broad proteins directly determines if a cell will Ark5 thus leads to ATP collapse and collaboration and knowledge exchange, with chromosomal amplification, gene undergo mitochondrial outer membrane consequently loss of viability, selectively in cells multiple one-to-one interactions ongoing on translocation, enhanced mRNA and protein permeabilisation (MOMP), widely considered overexpressing Myc, suggesting that targeting an ad hoc basis. stability or indeed increased signalling through to be a point of no return, in response to death these kinases may be therapeutically effective upstream regulatory factors such as Ras, Notch signalling. In a study that we published this year against Myc overexpressing cancers. We are Major developments in 2014 or β-catenin. In a number of in vivo settings, Myc in Cell Reports, we demonstrated a prominent using genetic models to investigate the We published our first independent work since overexpression is sufficient to initiate or role for the pro-apoptotic protein Bim during therapeutic potential of targeting Ark5 in our arrival in Glasgow in 2012 and collaborated exacerbate tumourigenesis and moreover is Myc-induced apoptosis in multiple settings. cancers that typically overexpress Myc, such on two other manuscripts that are presently typically required to sustain the cancerous Acute overexpression of Myc in the intestine as colorectal and pancreatic cancer. under review. Excellent progress was made in phenotype. A successful therapeutic strategy drives abundant apoptosis that is abrogated by our analysis of lung cancer progression and the that exploits Myc overexpression would likely deletion of Bim but unaffected by deletion of Myc-induced lung cancer progression in vivo requirement for Ark5 for colorectal have a tremendous impact on human health. either Puma (BBC3) or p19Arf. Conversely, Lung cancer remains one of the deadliest forms tumourigenesis. In collaboration with Owen apoptosis induced by the DNA damaging agent of cancer worldwide, accounting for some 18% Sansom and Jennifer Morton, we secured Bim mediates Myc-induced apoptosis doxorubicin requires Puma but not Bim. of all cancer-related deaths, and its incidence is funding from Worldwide Cancer Research to Expression of supra-physiological levels of Myc Strikingly, we found no requirement for p19Arf on the rise especially in the increasingly investigate the functional role of Myc in triggers apoptosis in otherwise non- during Myc-induced apoptosis under any of the industrialised and densely populated cities of pancreatic tumourigenesis. I served as co- transformed cells, and many tumour cells can conditions tested, suggesting that the major emerging economies. Poor prognosis arises in organiser of the Beatson International Cancer be induced to die upon forced expression of tumour suppressive activity of p19Arf derives large part from the combination of late disease Conference ‘Powering the Cancer Machine’ and exogenous Myc. A large number of publications from a non-apoptotic function. Encouragingly, detection and limited matching of patients with co-authored a subsequent meeting report, link Myc-driven apoptosis to induction of the these observations suggest the possibility of emerging targeted therapies. We have published in Cancer and Metabolism, with Jurre tumour suppressor p19Arf, followed by augmenting Myc’s pro-apoptotic signal, for developed a mouse model for early lung cancer Kamphorst. accumulation of p53, however Myc can also instance through the use of BH3 mimetics, even progression using tractable combinations of trigger apoptosis in the absence of a functional in cancers that lack functional p53. conditional alleles, including Kras and Myc. We Publications listed on page 88 p19-p53 pathway. Recent evidence has have used laser capture micro-dissection

68 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE ONCOGENE-INDUCED VULNERABILITIES 69 cell death mediated by the ubiquitin-like MITOCHONDRIA AND CELL DEATH properties of ATG12 (Fig. 2). www.beatson.gla.ac.uk/stephen_tait Mitochondria and non-apoptotic routes to cancer cell death Various other forms of programmed cell death exist besides apoptosis, including caspase- independent cell death, necroptosis and mitotic cell death. These alternate forms of cell death function as either back-up pathways in the event of failed apoptosis or are distinct cell death programmes in themselves. Although Cell death inhibits tumourigenesis at multiple stages, ranging these cell death modalities are likely important in cancer (particularly in response to therapy), from transformation to metastasis. Consequently, in order for little is known about their underlying molecular cancer to develop, cell death must be inhibited. Moreover, the mechanisms. Mitochondria have been implicated as major effectors in necroptosis, a best way to treat a cancer cell is to kill it, indeed many cancer Figure 1 the role of ATG12 in mitochondrial clearance, form of regulated cell death that requires therapies do just this. Therefore, cell death sensitivity also The ubiquitin-like protein ATG12 we found that the unconjugated, free form of activation of the kinase RIPK3. We have directly is itself targeted for ubiquitination. ATG12 is rapidly degraded in a proteasome- tested a role for mitochondria in necroptosis by affects how well anticancer therapy works. Mitochondria, the ATG12 was co-expressed with His-tagged ubiquitin in the dependent manner. Strikingly, ATG12, itself an generating mitochondria-free cells through cellular organelles that power life, are also essential for the presence or absence of ubiquitin-like protein, is directly targeted for Parkin-mediated mitophagy. Using this method, Group Leader proteasome inhibitor MG132. ubiquitination and this promotes its we find that the kinetics and extent of major form of programmed cell death called apoptosis. Our Left: ubiquitinated proteins were proteasome-dependent degradation (Fig. 1). As necroptosis are not affected by mitochondrial Stephen Tait pulled down and blotted with a functional consequence of this turnover, depletion, thereby ruling out a major role for Royal Society University overall aim is to understand how mitochondria regulate cell anti-ATG12 antibody revealing ATG12 contributes to proteasome-mediated cell mitochondria in executing necroptosis. Our Research Fellow death and define how this process is deregulated in cancer. an immunoreactive smear, Beatson Associate demonstrating direct ATG12 death - this may be clinically important given current studies focus upon investigating The ultimate goal is to clinically translate these findings to ubiquitination. Right: cell lysates the use of proteasome inhibitors as anticancer whether mitochondrial dysfunction can serve to Research Scientists were probed with ubiquitin or therapies. Our data develop an emerging initiate necroptosis, in addition to defining the ATG12 antibody showing an Martina Haller improve existing anticancer therapies and develop new paradigm that ubiquitin-like proteins themselves role for necroptosis in tumour suppression and 1 increase in ATG12 and Gabriel Ichim therapy-induced cell death. Maria Karvela means to selectively kill cancer cells. polyubiquitination levels can be directly modified by ubiquitin. Moreover, following proteasome inhibition. our results unveil a novel interconnection Graduate Student between autophagy, proteasome activity and Publications listed on page 88 Evangelos Giampazolias2 Mitochondria, cell death and cancer permeabilisation and apoptosis. In this method, Apoptosis requires caspase protease a destabilisation domain is fused to the Visitor activation leading to widespread substrate N-terminus of Bcl-XL, resulting in its constitutive Figure 2 ATG12 connects autophagy and Jonathan Lopez cleavage and rapid cell death. During degradation. Addition of stabilising ligand proteasome function to cell 1 EMBO apoptosis, mitochondrial outer membrane effectively upregulates Bcl-XL expression within death. Conjugation of ATG12 to 2 CRUK Glasgow Centre permeabilisation (MOMP) occurs, a crucial event a matter of minutes, causing cellular resistance ATG5 is required for autophagy. that is required for caspase activation. Following to MOMP and apoptosis. In collaboration with Free ATG12 is rapidly degraded by MOMP, mitochondrial inter-membrane space Frank Edlich (University of Freiburg), we find that the proteasome through ubiquitin-dependent and proteins, such as cytochrome c, are released switching on Bcl-XL expression leads to independent means. Stabilisation into the cytoplasm where they drive caspase shuttling of Bak, a key pro-apoptotic effector of ATG12, for example by activation and apoptosis. Given its key role in protein, from the mitochondria into the proteasome inhibition, controlling cell survival, mitochondrial outer cytoplasm. This demonstrates that so called promotes cell death. membrane integrity is highly regulated, largely retro-translocation is a conserved process for through interactions between pro- and anti- pro-apoptotic Bcl-2 proteins. Moreover, it apoptotic Bcl-2 proteins. Cancer cells highlights that enhancement of retro- commonly inhibit apoptosis by preventing translocation by anti-apoptotic Bcl-2 proteins MOMP, often through upregulation of anti- provides an additional level of protection against apoptotic Bcl-2 proteins or by inhibiting cell death. caspase activity downstream of MOMP. Newly developed anticancer therapies target these ATG12 connects autophagy and proteasome apoptotic blocks. For example, BH3 mimetic function to cell death compounds exploit the Bcl-2 addiction of Autophagic degradation and proteasome- certain cancer cells leading to tumour dependent degradation constitute the cell’s two specific killing. main protein degradation mechanisms. The ubiquitin-like protein ATG12 is an essential Switchable Bcl-XL provides new insight into autophagy protein. In an ubiquitination-like the commitment to die cascade, ATG12 is covalently conjugated to We have developed a new approach that ATG5 and the ATG12-5 conjugate is required for conditionally blocks mitochondrial autophagosome biogenesis. While investigating

70 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE MITOCHONDRIA AND CELL DEATH 71 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

73 RESEARCH FACILITIES

Research Facilities supports research groups at the Beatson Institute and University of Glasgow on the Beatson site. This year, there has been investment in new equipment for Molecular Technology, which replaced its Illumina GAIIx next generation sequencer with an Illumina NextSeq500 platform. This will enable the sequencing of libraries at a lower cost with increased data output and a faster turnaround time. Head of Research Facilities Building Facilities has been active with a number of projects to Sue Fowler refurbish essential services and adapt space to accommodate additional staff. allowing large scale consistent processing but can also be optimised to produce a working when required specialised processing cycles protocol that allows the antibody to be used Building Facilities glassware, sterilisation of consumables, and can be designed. Other material such as either on the autostainer or for hand staining by Alistair Wilson, Alex Kernahan, Michael Daly preparation of tissue culture solutions, bacterial organotypic assays, cell pellets, spheroids and the researcher. Training can be provided in order culture media and Drosophila food. The agar plugs can also be processed to provide a that an individual can understand the rationale Building Facilities manages the outsourced team is also responsible for the cleaning and wax block to allow sectioning and further and techniques available to allow them to services provision for catering, cleaning and checking of items such as centrifuge rotors, investigation. All paraffin wax blocks sectioned perform the staining to an acceptable and janitorial services. We provide maintenance X-ray processors, water baths and pH meters. are stained with haematoxylin and eosin in order consistent standard. support for the Beatson building and manage The stocking of the tissue culture suites, to allow general analysis of cell morphology alterations and refurbishments. This year there and laboratory waste collections and and structure. After initial analysis more Where there is no antibody available for have been two major projects to replace an autoclave processing to make waste safe specialised histology stains can be performed if immunohistochemistry analysis or a more ageing steam and chiller system in one of the are performed daily. required to investigate specific tissue structures. specific conclusive technique is required, the services areas. In addition, the ventilation in the If suitable material is available, mouse tissue service can provide an in situ hybridisation cryostore has been upgraded to increase the microarrays (TMAs) can also be constructed technique using a reagent system designed to number of air changes in the room. Histology Service using paraffin-embedded tissue. visualise cellular RNA targets in formalin-fixed, Colin Nixon, Brenda McGuire, Fiona McGregor, paraffin-embedded tissue sections using Use of the online helpdesk facility continues to Gemma Thomson, Mark Hughes, Saira Ghafoor, Where fixation is not required or bright-field microscopy. Specific probes can be be an effective means of logging reactive calls Vivienne Morrison, Wendy Lambie disadvantageous to tissue structure and purchased or designed to exact specifications for maintenance and repair. Minor project work analysis, the facility offers a frozen section by external companies such as Advanced Cell continues at a fairly high level. A number of The Histology Service performs essential resource. Frozen tissue, embryos or cells Diagnostics along with the necessary retrieval building services have been altered or extended processing of tissue samples and cellular can be sectioned and when required and amplification kits, allowing the in situ to accommodate new equipment. This year, material from the wide range of cancer models stained for examination using routine technique to be performed. fixed wire installation electrical testing was developed within the Institute allowing the immunohistochemical or immunofluorescence completed across the buildings. material to be evaluated at a cellular level in staining methods. Material for DNA/RNA The Institute has a Leica LMD6500 laser order to understand the disease mechanics. The investigation, PCR analysis and microdissection system that allows service offers processing for tissue samples immunofluorescence staining can also be subpopulations of tissue cells to be procured Central Services fixed in an array of different types of fixative sectioned from both paraffin-embedded from histological prepared slides under Margaret Laing (Supervisor), Elizabeth dependent on required subsequent analysis. material and frozen tissue. microscopic visualisation. We are able to cut Cheetham, Barbara Donnelly, Dilhani Kahawela, Once received the tissue samples will be sections from both cryostat and paraffin blocks Barbara Lambie, Kirstie McPherson, Tracy trimmed, appropriately processed and then A comprehensive immunohistochemistry onto specialised slides, which can be stained Shields, Rose Steel, Robert Storey orientated into paraffin wax blocks to facilitate service is offered using our two large capacity appropriately allowing cellular material to be tissue sectioning and staining. The tissue autostainers. We are continually expanding the identified and separated to permit subsequent Central Services performs a wide range of duties samples are processed according to type and number of optimised antibodies that, if required, downstream analysis to be performed. Both that are essential for the support of the research necessity using previously designated specific, can be batch-stained using the facility’s DNA and RNA material can be retrieved from the groups across the site. This includes cleaning specialised processing cycles. We have three immunohistochemistry autostainers to provide tissue sections for downstream analysis. and sterilisation of reusable laboratory large capacity automated tissue processors high quality, consistent staining. New antibodies

74 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH FACILITIES 75 A fully automated large capacity Leica SCN400F the foundation of our business continuity equipment to facilitate the needs of researchers. Small scale DNA purification is performed on a slide scanner has been installed in the facility, documentation and has led to the revision and The servicing and maintenance of core Qiagen 8000 Biorobot. Researchers provide which is capable of capturing bright-field or ongoing improvement of many of our equipment and any systems that these require, overnight bacterial cultures that are processed fluorescent images. This allows high quality day-to-day working practices. such as carbon dioxide or nitrogen gas, is by the facility. Sample numbers are consistently digital images to be scanned, stored and, if carefully managed and coordinated to ensure in the region of 15,000-17,000 per year. We required, quantitative interpretation performed. Our intranet uses a content management equipment breakdowns are kept to a minimum. continue to provide a very popular large scale The image analysis software allows staining system (CMS) framework, allowing service Any equipment repairs are coordinated to DNA purification (maxiprep) service from techniques to be scored using algorithms managers and support departments the ability ensure these are dealt with as efficiently and bacterial cultures. designed specifically for that staining, using the to easily upload forms and information for effectively as possible. Service contracts for researchers input to designate what specific users. Bespoke hardware systems have been core equipment are reviewed and procured Human cell line authentication using the areas are to be scored. designed and configured for users allowing centrally to ensure costs are kept as low as Promega Geneprint 10 Kit is available as an them to achieve significant speed gains when reasonably practical. We have effective internal service. The samples are run on the they are running large data analyses, in some procurement processes and liaise with Applied Biosystems 3130xl Sequencer (Gene Information Services cases reducing data analysis runs from days Cancer Research UK Purchasing to take Fragment Analysis) and analysed using Peter McHardy, Iain White to minutes. advantage of any centralised agreements. A Genemapper v4.0 software (Applied further essential role is the monitoring of all Biosystems). Regular cell line authentication is Information Services provides a wide range of A range of replacement hardware is stored on outgoing orders to ensure compliance with important both to confirm integrity of data and support services, including server support, site to allow fast repairs. A good selection of Institute safety procedures, particularly is increasingly requested by journals as a hardware cover, an on-site helpdesk providing loan IT hardware, from USB drives to digital those relating to COSHH. requirement prior to publication both repair and software support as well as help projectors, is held centrally. We provide video in hardware selection and user training. There conferencing facilities, enabling conference The stores facility stocks a wide range of Reagent Services provide a diverse range of are over 350 users with nearly 400 PCs on site calls between the Beatson and other Cancer consumables with rapid re-stocking to ensure support to the research groups. The comprising a mixture of Windows computers, Research UK sites as well as many other high use materials are always available. Items mycoplasma screening service offers testing of Apple Macs and Linux machines, with central locations. Audio-visual support services for can be withdrawn on a self-service basis with each research group’s cells every four to five authentication, central file store and print large conferences have been provided at a automatic cost centre allocation via swipe card. months. Researchers are also encouraged to sharing. The servers provide in excess of 500 TB number of international venues, as well as A porter service is run to deliver external orders have newly imported cell lines tested as soon as of online storage with nightly backups and tapes overseeing the in-house 178-seat state- to the researchers, while outgoing samples or possible after arrival as we have found that a stored off-site, to provide support for of-the-art lecture theatre. materials are processed by stores for courier significant number of newly imported cell lines microscopy, DNA sequencing and mass collection. We continue to review the services are infected with mycoplasma. Cell lines are spectrometry data. provided by stores to improve what is offered to mainly tested using a luciferase assay that Laboratory Management scientific staff. This includes negotiating free detects mycoplasmal enzymes. They may also All PCs are built with a common desktop Laura Bence, Richard Selkirk, Michael samples from suppliers to enable the scientific be tested by: Hoechst staining to detect the environment, around Windows or Mac OS X and McTaggart, Joe McFadden, George Monteith staff to assess new or alternative products. By presence of mycoplasma DNA; enzyme Microsoft Office, and are actively managed and maintaining a good relationship with suppliers immunoassay against the four most common upgraded to ensure the best possible working Laboratory Management is responsible for preferential pricing is obtained and, as a result of species of mycoplasma; or a colorimetric environment. Mac OS X Yosemite is being rolled providing a number of vital support roles to the these negotiations and better turnaround times microplate assay to detect 16S ribosomal out across the site and we have completed the Institute. This includes the provision of advice, from suppliers, we have been able to reduce mycoplasma RNA. upgrade of relevant Windows computers to training and information to all staff on health the overall value of stock held without Windows 7. All e-mail services run on Microsoft and safety issues, especially with regard to risk compromising supply lines to the laboratories. Cell-derived matrices from Tiff 5 cells are Exchange, which allows local client-based assessments and appropriate control measures prepared to order for the research groups and access and web access to email as well as necessary for laboratory work involving have proved very popular. Stocks of commonly delivering email, diaries and address books to biological, chemical and genetic modification Molecular Technology and Reagent Services used tissue culture medium are ordered and the mobile devices including iPhones, iPads and processes. Safety in regard to fire risk is also Billy Clark, Deborah Gardner, Andrew Keith batch testing of serum is coordinated. The other smart phones. managed. As safety plays an important part of facility provides a range of commonly used everyday life in the laboratory, and in running The Molecular Technology Service provides buffers, for example 10X TBST and bacterial We continue to migrate over as many physical building services, it is essential that health and routine plasmid sequencing and DNA growth reagents. Each product is tested for servers as possible to virtual servers using safety processes are reviewed and monitored purification on a small and large scale. suitability of use and sterility where possible VMware®. We provide access to virtualised regularly, that any training needs are rectified Sequencing is performed on an Applied before being released for general stock. The servers for research groups allowing them and that adequate provision is made to fulfil the Biosystems 3130xl (16 capillary) Sequencer that preparation of antibiotic bacterial culture plates greater flexibility for both test and production Institute’s legal obligations to staff. All staff and provides good sample throughput, long read has been automated using a Mediaclave (Integra applications. This also allows us to provide students are required to attend a safety update lengths and a sample turnaround time of 24 Biosciences AG) to sterilise and dispense into virtual workstations for researchers with both seminar once a year and new starts attend a hours. In recent years, DNA sequencing has the plates. high core counts and large amounts of RAM, series of safety induction talks. This year some been revolutionised by the introduction of next making them ideal for mass spectrometry of the first aiders were trained in oxygen generation technologies offering large scale analysis or other computationally intense administration and a defibrillator will be sequencing in a matter of hours. This year, we applications. We currently provide virtual acquired soon for use within the Institute. In replaced our Illumina GAIIx with an Illumina desktops for OS X users requiring access to addition, a new risk assessment form has NextSeq500 platform. This will enable us to Windows-based packages. been created to include the new harmonised sequence libraries at a lower cost with increased hazard codes. data output and a faster turnaround time. We Significant investment has been put into also offer library production for next generation providing documentation and procedures to A major function of Laboratory Management is sequencing. Protocols currently in use are allow us to run the service in a manner the overseeing of shared equipment servicing, ChIP-seq and RNA-seq. commensurate with ITIL. This has been used as replacement and the purchase of new

76 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH FACILITIES 77 Li A, Morton JP, Ma Y, Karim SA, Zhou Y, Faller WJ, Arjonen A, Kaukonen R, Mattila E, Rouhi P, Hognas RESEARCH PUBLICATIONS Woodham EF, Morris HT, Stevenson RP, Juin A, G, Sihto H, Miller BW, Morton JP, Bucher E, Taimen Jamieson NB, MacKay CJ, Carter CR, Leung HY, P, Virtakoivu R, Cao Y, Sansom OJ, Joensuu H, Yamashiro S, Blyth K, Sansom OJ, Machesky LM. Ivaska J. Fascin is regulated by slug, promotes progression of Mutant p53-associated myosin-X upregulation pancreatic cancer in mice, and is associated with promotes breast cancer invasion and metastasis. J patient outcomes. Gastroenterology 2014; 146: Clin Invest 2014; 124: 1069-82 1386-96 e1-17 Cassier PA, Polivka V, Judson I, Soria JC, Penel N, McDonald L, Ferrari N, Terry A, Bell M, Mohammed Marsoni S, Verweij J, Schellens JH, Morales- ZM, Orange C, Jenkins A, Muller WJ, Gusterson BA, Barrera R, Schoffski P, Voest EE, Gomez-Roca C, Neil JC, Edwards J, Morris JS, Cameron ER, Blyth K. Evans TR, Plummer R, Gallerani E, Kaye SB, Olmos Kurt Anderson (page 26) Pajak MZ, Oien KA, McKay CJ, Carter CR, Gillen G, RUNX2 correlates with subtype-specific breast D. Outcome of patients with sarcoma and other Tumour Cell Migration Champion S, Pimlott SL, Anderson KI, Evans TR, cancer in a human tissue microarray, and ectopic mesenchymal tumours participating in phase I Grimmond SM, Biankin AV, Sansom OJ, Morton JP. expression of Runx2 perturbs differentiation in the trials: a subset analysis of a European Phase I Targeting mTOR dependency in pancreatic cancer. mouse mammary gland. 2014; database. 2014; 25: 1222-8 Primary Research Papers Dis Model Mech Ann Oncol Gut 2014; 63: 1481-9 7: 525-34 Ioannidou K, Anderson KI, Strachan D, Edgar JM, Faller WJ, Jackson TJ, Knight JR, Ridgway RA, Barnett SC. Rai TS, Cole JJ, Nelson DM, Dikovskaya D, Faller W, Rai TS, Cole JJ, Nelson DM, Dikovskaya D, Faller W, Jamieson T, Karim SA, Jones C, Radulescu S, Huels Astroglial-axonal interactions during early stages Vizioli MG, Hewitt RN, Anannya O, McBryan T, Vizioli MG, Hewitt RN, Anannya O, McBryan T, DJ, Myant KB, Dudek KM, Casey HA, Scopelliti A, of myelination in mixed cultures using in vitro Manoharan I, van Tuyn J, Morrice N, Pchelintsev Manoharan I, van Tuyn J, Morrice N, Pchelintsev Cordero JB, Vidal M, Pende M, Ryazanov AG, and ex vivo imaging techniques. BMC Neurosci NA, Ivanov A, Brock C, Drotar M, Nixon C, NA, Ivanov A, Brock C, Drotar M, Nixon C, Sonenberg N, Meyuhas O, Hall MN, Bushell M, 2014; 15: 59 Clark W, Sansom OJ, Anderson KI, King A, Clark W, Sansom OJ, Anderson KI, King A, Blyth K, Willis AE, Sansom OJ. Blyth K, Adams PD. Adams PD. mTORC1-mediated translational elongation limits Johnsson AK, Dai Y, Nobis M, Baker MJ, McGhee HIRA orchestrates a dynamic chromatin landscape HIRA orchestrates a dynamic chromatin landscape intestinal tumour initiation and growth. EJ, Walker S, Schwarz JP, Kadir S, Morton JP, Myant Nature in senescence and is required for suppression of in senescence and is required for suppression of 2015; 517: 497-500, doi: 10.1038/nature13896. Epub KB, Huels DJ, Segonds-Pichon A, Sansom OJ, neoplasia. Genes Dev 2014; 28: 2712-25 neoplasia. 2014; 28: 2712-25 2014 Nov 5 Anderson KI, Timpson P, Welch HC. Genes Dev The Rac-FRET mouse reveals tight spatiotemporal Vandenberg CJ, Josefsson EC, Campbell KJ, Hamilton G, Abraham AG, Morton J, Sampson O, control of Rac activity in primary cells and tissues. James C, Lawlor KE, Kile BT, Cory S. Pefani DE, Khoronenkova S, Grawenda A, Cell Rep 2014; 6: 1153-64 Karen Blyth (page 58) Transgenic Models of Cancer Loss of Bak enhances lymphocytosis but does not Papaspyropoulos A, Jamieson N, McKay C, ameliorate thrombocyteppaenia in BCL-2 Sansom O, Dianov GL, O’Neill E. Lo Re D, Zhou Y, Nobis M, Anderson KI, Murphy PV. transgenic mice. 2014; 21: 676-84 AKT regulates NPM dependent ARF localization and Synthesis of migrastatin and its macroketone Primary Research Papers Cell Death Differ p53mut stability in tumors. 2014; 5: analogue and in vivo FRAP analysis of the Ferrari N, Mohammed ZM, Nixon C, Mason SM, Oncotarget 6142-67 macroketone on E-cadherin dynamics. Mallon E, McMillan DC, Morris JS, Cameron ER, Chembiochem 2014; 15: 1459-64 Edwards J, Blyth K. Martin Drysdale (page 48) Herrmann D, Conway JR, Vennin C, Magenau A, Expression of RUNX1 correlates with poor patient Drug Discovery Programme Hughes WE, Morton JP, Timpson P. Morran DC, Wu J, Jamieson NB, Mrowinska A, prognosis in triple negative breast cancer. PLoS One Three-dimensional cancer models mimic cell- Kalna G, Karim SA, Au AY, Scarlett CJ, Chang DK, 2014; 9: e100759 Primary Research Papers matrix interactions in the tumour Unbekandt M, Croft DR, Crighton D, Mezna M, microenvironment. Carcinogenesis 2014; 35: 1671-9 McArthur D, McConnell P, Schuttelkopf AW, Belshaw S, Pannifer A, Sime M, Bower J, Drysdale Johnsson AK, Dai Y, Nobis M, Baker MJ, McGhee M, Olson MF. EJ, Walker S, Schwarz JP, Kadir S, Morton JP, Myant A novel small-molecule MRCK inhibitor blocks KB, Huels DJ, Segonds-Pichon A, Sansom OJ, cancer cell invasion. Cell Commun Signal 2014; Anderson KI, Timpson P, Welch HC. 12: 54 The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Rep 2014; 6: 1153-64 Jeff Evans (page 28) Translational Cancer Therapeutics Li A, Morton JP, Ma Y, Karim SA, Zhou Y, Faller WJ, Woodham EF, Morris HT, Stevenson RP, Juin A, Jamieson NB, MacKay CJ, Carter CR, Leung HY, Primary Research Papers Yamashiro S, Blyth K, Sansom OJ, Machesky LM. Ali A, Brown V, Denley S, Jamieson NB, Morton JP, Fascin is regulated by slug, promotes progression of Nixon C, Graham JS, Sansom OJ, Carter CR, pancreatic cancer in mice, and is associated with McKay CJ, Duthie FR, Oien KA. patient outcomes. 2014; 146: Expression of KOC, S100P, mesothelin and MUC1 Gastroenterology 1386-96 e1-17 in pancreatico-biliary adenocarcinomas: development and utility of a potential diagnostic Middleton G, Silcocks P, Cox T, Valle J, Wadsley J, immunohistochemistry panel. BMC Clin Pathol Propper D, Coxon F, Ross P, Madhusudan S, 2014; 14: 35 Roques T, Cunningham D, Falk S, Wadd N, Harrison

78 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 79 M, Corrie P, Iveson T, Robinson A, McAdam K, Eyal Gottlieb (page 12) Robert Insall (page 30) Grimmond SM, Biankin AV, Sansom OJ, Morton JP. Eatock M, Evans J, Archer C, Hickish T, Garcia- Tumour Metabolism Cell Migration and Chemotaxis Targeting mTOR dependency in pancreatic cancer. Alonso A, Nicolson M, Steward W, Anthoney A, Gut 2014; 63: 1481-9 Greenhalf W, Shaw V, Costello E, Naisbitt D, Primary Research Papers Primary Research Papers Rawcliffe C, Nanson G, Neoptolemos J. 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Dose-response relationship in phase I A, Geiger T, Clemons PA, Gottlieb E, Ruppin E. Primary Research Papers clinical trials: A European drug development Predicting cancer-specific vulnerability via data- Foth M, Ahmad I, van Rhijn BW, van der Kwast T, network (Eddn) collaboration study. Clin Cancer Res driven detection of synthetic lethality. 2014; Bergman AM, King L, Ridgway R, Leung HY, Fraser 2014; 20: 5663-71 Cell Shehab Ismail (page 32) 158: 1199-209 Structural Biology of Cilia S, Sansom OJ, Iwata T. Fibroblast growth factor receptor 3 activation plays a causative role in Morran DC, Wu J, Jamieson NB, Mrowinska A, Oburoglu L, Tardito S, Fritz V, de Barros SC, Merida urothelial cancer pathogenesis in cooperation with Kalna G, Karim SA, Au AY, Scarlett CJ, Chang DK, Primary Research Papers P, Craveiro M, Mamede J, Cretenet G, Mongellaz C, Pten loss in mice. J Pathol 2014; 233: 148-58 Pajak MZ, Oien KA, McKay CJ, Carter CR, Gillen G, Bravo-Rodriguez K, Ismail-Ali AF, Klopries S, An X, Klysz D, Touhami J, Boyer-Clavel M, Battini Champion S, Pimlott SL, Anderson KI, Evans TR, Kushnir S, Ismail S, Fansa EK, Wittinghofer A, Schulz JL, Dardalhon V, Zimmermann VS, Mohandas N, Li A, Morton JP, Ma Y, Karim SA, Zhou Y, Faller WJ, Grimmond SM, Biankin AV, Sansom OJ, Morton JP. F, Sanchez-Garcia E. Predicted incorporation of Gottlieb E, Sitbon M, Kinet S, Taylor N. Glucose and Woodham EF, Morris HT, Stevenson RP, Juin A, Targeting mTOR dependency in pancreatic cancer. non-native substrates by a polyketide synthase glutamine metabolism regulate human Jamieson NB, MacKay CJ, Carter CR, Leung HY, Gut 2014; 63: 1481-9 yields bioactive natural product derivatives. hematopoietic stem cell lineage specification. Cell Yamashiro S, Blyth K, Sansom OJ, Machesky LM. 2014; 15: 1991-7 2014; 15: 169-84 Chembiochem Fascin is regulated by slug, promotes progression of Puzanov I, Lindsay CR, Goff LW, Sosman JA, Gilbert Stem Cell pancreatic cancer in mice, and is associated with J, Berlin J, Poondru S, Simantov R, Gedrich R, Suladze S, Ismail S, Winter R. Thermodynamic, Witney TH, Pisaneschi F, Alam IS, Trousil S, patient outcomes. Gastroenterology 2014; 146: Stephens A, Chan E, Evans TR. dynamic and solvational properties of PDEdelta Kaliszczak M, Twyman F, Brickute D, Nguyen QD, 1386-96 e1-17 A phase I study of continuous oral dosing of binding to farnesylated cystein: a model study for Schug Z, Gottlieb E, Aboagye EO. Preclinical OSI-906, a dual inhibitor of insulin-like growth uncovering the molecular mechanism of PDEdelta evaluation of 3-18F-fluoro-2,2-dimethylpropionic Mardilovich K, Gabrielsen M, McGarry L, Orange C, factor-1 and insulin receptors in patients with interaction with prenylated proteins. acid as an imaging agent for tumor detection. J Nucl J Phys Chem B Patel R, Shanks E, Edwards J, Olson MF. Elevated advanced solid tumors. Clin Cancer Res 2015; 21: 2014; 118: 966-75 2014; 55: 1506-12 LIM kinase 1 in non-metastatic prostate cancer 701-11. doi: 10.1158/1078-0432.CCR-14-0303. Epub Med reflects its role in facilitating androgen receptor 2014 Sep 11 Zimmermann G, Schultz-Fademrecht C, Kuchler P, nuclear translocation. Mol Cancer Ther 2015; 14: Murarka S, Ismail S, Triola G, Nussbaumer P, 246-58. doi: 10.1158/1535-7163.MCT-14-0447. Roxburgh P, Lumsden GR, Paul J, Harden S, Wittinghofer A, Waldmann H. Structure guided Epub 2014 Oct 24 Sweeting L, James A, Crellin A, Morrison R, Evans design and kinetic analysis of highly potent TR, McDonald AC. benzimidazole inhibitors targeting the PDEdelta Munkley J, Rajan P, Lafferty NP, Dalgliesh C, A phase I and pharmacokinetic study of prenyl binding site. 2014; 57: 5435-48 J Med Chem Jackson RM, Robson CN, Leung HY, Elliott DJ. A capecitabine in combination with radiotherapy in novel androgen-regulated isoform of the TSC2 patients with localised inoperable pancreatic tumour suppressor gene increases cell cancer. Cancer Chemother Pharmacol 2014; (page 53) proliferation. 2014; 5: 131-9 74: 131-9 Gabriela Kalna Oncotarget Bioinformatics Noble-Jones R, Fitzpatrick B, Sneddon MC, Zhu AX, Rosmorduc O, Evans TR, Ross PJ, Santoro Hendry DS, Leung HY. Development of the A, Carrilho FJ, Bruix J2, Qin S, Thuluvath PJ, Llovet Primary Research Papers lymphoedema genito-urinary cancer JM, Leberre MA, Jensen M, Meinhardt G, Kang YK. 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80 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 81 Gillian Mackay (page 54) Metabolomics Rainero E, Cianflone C, Porporato PE, Chianale F, Malacarne V, Bettio V, Ruffo E, Ferrara M, Benecchia F, Capello D, Paster W, Locatelli I, Primary Research Papers Bertoni A, Filigheddu N, Sinigaglia F, Norman JC, Labuschagne CF, van den Broek NJ, Mackay GM, Baldanzi G, Graziani A. The diacylglycerol kinase Vousden KH, Maddocks OD. Serine, but not alpha/atypical PKC/beta1 integrin pathway in glycine, supports one-carbon metabolism and SDF-1alpha mammary carcinoma invasiveness. proliferation of cancer cells. 2014; 7: Cell Rep PLoS One 2014; 9: e97144 1248-58

Michael Olson (page 38) (page 55) Gaurav Malviya Molecular Cell Biology Nuclear Imaging

Primary Research Papers Primary Research Papers Mardilovich K, Gabrielsen M, McGarry L, Orange C, Morran DC, Wu J, Jamieson NB, Mrowinska A, Patel R, Shanks E, Edwards J, Olson MF. Elevated Kalna G, Karim SA, Au AY, Scarlett CJ, Chang DK, LIM kinase 1 in non-metastatic prostate cancer Pajak MZ, Oien KA, McKay CJ, Carter CR, Gillen G, reflects its role in facilitating androgen receptor Champion S, Pimlott SL, Anderson KI, Evans TR, nuclear translocation. Mol Cancer Ther 2015; 14: Grimmond SM, Biankin AV, Sansom OJ, Morton JP. 246-58. doi: 10.1158/1535-7163.MCT-14-0447. Targeting mTOR dependency in pancreatic cancer. Epub 2014 Oct 24 Gut 2014; 63: 1481-9 Prater MD, Petit V, Alasdair Russell I, Giraddi RR, Stockley J, Villasevil ME, Nixon C, Ahmad I, Leung Jamieson NB, MacKay CJ, Carter CR, Leung HY, Other Publications Shehata M, Menon S, Schulte R, Kalajzic I, Rath N, HY, Rajan P. The RNA-binding protein hnRNPA2 Yamashiro S, Blyth K, Sansom OJ, Machesky LM. Malviya G, Galli F, Sonni I, Signore A. Imaging Olson MF, Metzger D, Faraldo MM, Deugnier MA, regulates beta-catenin protein expression and is Fascin is regulated by slug, promotes progression of T-lymphocytes in inflammatory diseases: a nuclear Glukhova MA, Stingl J. Mammary stem cells have overexpressed in prostate cancer. RNA Biol 2014; 11: pancreatic cancer in mice, and is associated with medicine approach. Q J Nucl Med Mol Imaging myoepithelial cell properties. Nat Cell Biol 2014; 16: 755-65 patient outcomes. Gastroenterology 2014; 146: 2014; 58: 237-57 942-50 1386-96 e1-17 Other Publications Malviya G, Signore A. Infection and inflammation Unbekandt M, Croft DR, Crighton D, Mezna M, Mukherjee A, Morton S, Fraser S, Salmond J, Baxter Lindsay CR, Li A, Faller W, Ozanne B, Welch H, imaging. 2014; 41: 488 Nucl Med Biol McArthur D, McConnell P, Schuttelkopf AW, G, Leung HY. Magnetic resonance imaging- Machesky LM, Sansom OJ. A Rac1-independent Belshaw S, Pannifer A, Sime M, Bower J, Drysdale directed transperineal limited-mapping prostatic role for P-Rex1 in melanoblasts. J Invest Dermatol Signore A, de Vries EFJ, Galli F, Malviya G. M, Olson MF. A novel small-molecule MRCK biopsies to diagnose prostate cancer: a Scottish 2015; 135: 314-8. doi: 10.1038/jid.2014.323. Epub Applications of molecular small-animal imaging in inhibitor blocks cancer cell invasion. Cell Commun experience. Scott Med J 2014; 59: 204-8 2014 Jul 30 inflammation and infection. In: Molecular Imaging Signal 2014; 12: 54 of Small Animals: Instrumentation and Applications. Muinonen-Martin AJ, Susanto O, Zhang Q, Springer, New York, USA (ISBN: 9781493908943) Sergio Lilla & David Sumpton Other Publications Smethurst E, Faller WJ, Veltman DM, Kalna G, (page 56) Julian L, Olson MF. Rho-associated coiled-coil Lindsay C, Bennett DC, Sansom OJ, Herd R, Jones Proteomics and Mass Spectrometry containing kinases (ROCK): structure, regulation, R, Machesky LM, Wakelam MJ, Knecht DA, Insall Jim Norman (page 36) and functions. 2014; 5: e29846 RH. Melanoma cells break down LPA to establish Small GTPases Primary Research Papers Integrin Cell Biology local gradients that drive chemotactic dispersal. Vijayakumar V, Monypenny J, Chen XJ, Unbekandt M, Olson MF. The actin-myosin Machesky LM, Lilla S, Thrasher AJ, Anton IM, Calle PLoS Biol 2014; 12: e1001966 Primary Research Papers regulatory MRCK kinases: regulation, biological Y, Jones GE. functions and associations with human cancer. Vijayakumar V, Monypenny J, Chen XJ, Machesky Haugsten EM, Brech A, Liestol K, Norman JC, J Tyrosine phosphorylation of WIP releases bound (Berl) 2014; 92: 217-25 LM, Lilla S, Thrasher AJ, Anton IM, Calle Y, Jones Wesche J. Photoactivation approaches reveal a role Mol Med WASP and impairs podosome assembly in GE. Tyrosine phosphorylation of WIP releases for Rab11 in FGFR4 recycling and signalling. Traffic macrophages. 2015; 128: 251-65. doi: J Cell Sci bound WASP and impairs podosome assembly in 2014; 15: 665-83 10.1242/jcs.154880. Epub 2014 Nov 20 macrophages. J Cell Sci 2015; 128: 251-65. doi: Kevin Ryan (page 18) 10.1242/jcs.154880. Epub 2014 Nov 20 Macagno JP, Diaz Vera J, Yu Y, MacPherson I, Tumour Cell Death Sandilands E, Palmer R, Norman JC, Frame M, Vidal M. FAK acts as a suppressor of RTK-MAP Laura Machesky (page 34) Other Publications Primary Research Papers kinase signalling in Drosophila melanogaster Migration, Invasion and Metastasis Ma Y, Machesky LM. Fascin1 in carcinomas: Its Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM. epithelia and human cancer cells. 2014; regulation and prognostic value. Int J Cancer 2014 PLoS Genet Using enhanced-mitophagy to measure Oct 10. doi: 10.1002/ijc.29260. [Epub ahead of print] 10: e1004262 Primary Research Papers autophagic flux. Methods 2015; 75: 105-11. doi: Garcia E, Machesky LM, Jones GE, Anton IM. WIP is 10.1016/j.ymeth.2014.11.014. Epub 2014 Dec 9 Machesky L, Sedwick C. Laura Machesky: actin Macpherson IR, Rainero E, Mitchell LE, van den necessary for matrix invasion by breast cancer cells. Berghe PV, Speirs C, Dozynkiewicz MA, Chaudhary opens the way. J Cell Biol 2014; 206: 816-7 Haller M, Hock AK, Giampazolias E, Oberst A, Eur J Cell Biol 2014; 93: 413-23 S, Kalna G, Edwards J, Timpson P, Norman JC. Green DR, Debnath J, Ryan KM, Vousden KH, Tait CLIC3 controls recycling of late endosomal Woodham EF, Machesky LM. Polarised cell SWG. Ubiquitination and proteasomal degradation Li A, Morton JP, Ma Y, Karim SA, Zhou Y, Faller WJ, MT1-MMP and dictates invasion and metastasis in migration: intrinsic and extrinsic drivers. Curr Opin of ATG12 regulates its pro-apoptotic activity, Woodham EF, Morris HT, Stevenson RP, Juin A, breast cancer. 2014; 127: 3893-901 Cell Biol 2014; 30C: 25-32 J Cell Sci Autophagy 2014; 10: 2269-78

82 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 83 Rosenfeldt MT, Bell LA, Long JS, O’Prey J, Nixon C, initiation and growth. Nature 2015; 517: 497-500. Roberts F, Dufes C, Ryan KM. E2F1 drives doi: 10.1038/nature13896. Epub 2014 Nov 5 chemotherapeutic drug resistance via ABCG2. Oncogene 2014; 33: 4164-72 Foth M, Ahmad I, van Rhijn BW, van der Kwast T, Bergman AM, King L, Ridgway R, Leung HY, Fraser Other Publications S, Sansom OJ, Iwata T. Fibroblast growth factor Joshi S, Ryan KM. Autophagy chews Fap to receptor 3 activation plays a causative role in promote apoptosis. Nat Cell Biol 2014; 16: 23-5 urothelial cancer pathogenesis in cooperation with Pten loss in mice. J Pathol 2014; 233: 148-58

Owen Sansom (page 40) Hamilton G, Abraham AG, Morton J, Sampson O, Colorectal Cancer and Wnt Signalling Pefani DE, Khoronenkova S, Grawenda A, Papaspyropoulos A, Jamieson N, McKay C, Sansom O, Dianov GL, O’Neill E. AKT regulates Primary Research Papers NPM dependent ARF localization and p53mut Ali A, Brown V, Denley S, Jamieson NB, Morton JP, stability in tumors. Oncotarget 2014; 5: 6142-67 Nixon C, Graham JS, Sansom OJ, Carter CR, McKay CJ, Duthie FR, Oien KA. Expression of KOC, Johnsson AK, Dai Y, Nobis M, Baker MJ, McGhee S100P, mesothelin and MUC1 in pancreatico-biliary EJ, Walker S, Schwarz JP, Kadir S, Morton JP, Myant adenocarcinomas: development and utility of a KB, Huels DJ, Segonds-Pichon A, Sansom OJ, potential diagnostic immunohistochemistry panel. Anderson KI, Timpson P, Welch HC. The Rac-FRET BMC Clin Pathol 2014; 14: 35 mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Rep 2014; 6: Arjonen A, Kaukonen R, Mattila E, Rouhi P, Hognas 1153-64 Muinonen-Martin AJ, Susanto O, Zhang Q, Scopelliti A, Cordero JB, Diao F, Strathdee K, White G, Sihto H, Miller BW, Morton JP, Bucher E, Taimen Smethurst E, Faller WJ, Veltman DM, Kalna G, BH, Sansom OJ, Vidal M. Local control of intestinal P, Virtakoivu R, Cao Y, Sansom OJ, Joensuu H, Li A, Morton JP, Ma Y, Karim SA, Zhou Y, Faller WJ, Lindsay C, Bennett DC, Sansom OJ, Herd R, Jones stem cell homeostasis by enteroendocrine cells in Ivaska J. Mutant p53-associated myosin-X Woodham EF, Morris HT, Stevenson RP, Juin A, R, Machesky LM, Wakelam MJ, Knecht DA, Insall the adult Drosophila midgut. Curr Biol 2014; 24: upregulation promotes breast cancer invasion and Jamieson NB, MacKay CJ, Carter CR, Leung HY, RH. Melanoma cells break down LPA to establish 1199-211 metastasis. J Clin Invest 2014; 124: 1069-82 Yamashiro S, Blyth K, Sansom OJ, Machesky LM. local gradients that drive chemotactic dispersal. Fascin is regulated by slug, promotes progression of PLoS Biol 2014; 12: e1001966 Song JH, Huels DJ, Ridgway RA, Sansom OJ, Belmont PJ, Budinska E, Jiang P, Sinnamon MJ, pancreatic cancer in mice, and is associated with Kholodenko BN, Kolch W, Cho KH. The APC Coffee E, Roper J, Xie T, Rejto PA, Derkits S, patient outcomes. Gastroenterology 2014; 146: Peter S, Bultinck J, Myant K, Jaenicke LA, Walz S, network regulates the removal of mutated cells Sansom OJ, Delorenzi M, Tejpar S, Hung KE, Martin 1386-96 e1-17 Muller J, Gmachl M, Treu M, Boehmelt G, Ade CP, from colonic crypts. Cell Rep 2014; 7: 94-103 ES. Cross-species analysis of genetically Schmitz W, Wiegering A, Otto C, Popov N, Sansom engineered mouse models of MAPK-driven Lindsay CR, Li A, Faller W, Ozanne B, Welch H, O, Kraut N, Eilers M. Tumor cell-specific inhibition Ueberham E, Glockner P, Gohler C, Straub BK, colorectal cancer identifies hallmarks of the human Machesky LM, Sansom OJ. A Rac1-independent of MYC function using small molecule inhibitors of Teupser D, Schonig K, Braeuning A, Hohn AK, disease. Dis Model Mech 2014; 7: 613-23 role for P-Rex1 in melanoblasts. J Invest Dermatol the HUWE1 ubiquitin ligase. EMBO Mol Med 2014; Jerchow B, Birchmeier W, Gaunitz F, Arendt T, 2015; 135: 314-8. doi: 10.1038/jid.2014.323. Epub 6: 1525-41 Sansom O, Gebhardt R, Ueberham U. Global Cordero JB, Ridgway RA, Valeri N, Nixon C, Frame 2014 Jul 30 increase of p16INK4a in APC-deficient mouse liver MC, Muller WJ, Vidal M, Sansom OJ. c-Src drives Phesse TJ, Myant KB, Cole AM, Ridgway RA, drives clonal growth of p16INK4a negative tumors. intestinal regeneration and transformation. EMBO J Lunardi S, Jamieson NB, Lim SY, Griffiths KL, Pearson H, Muncan V, van den Brink GR, Vousden Mol Cancer Res 2015 F; 13: 239-49. doi: 2014; 33: 1474-91 Carvalho-Gaspar M, Al-Assar O, Yameen S, Carter KH, Sears R, Vassilev LT, Clarke AR, Sansom OJ. 10.1158/1541-7786.MCR-14-0278-T. Epub 2014 RC, McKay CJ, Spoletini G, D’Ugo S, Silva MA, Endogenous c-Myc is essential for p53-induced Sep 30 Davis H, Irshad S, Bansal M, Rafferty H, Boitsova T, Sansom OJ, Janssen KP, Muschel RJ, Brunner TB. apoptosis in response to DNA damage in vivo. Cell Bardella C, Jaeger E, Lewis A, Freeman-Mills L, IP-10/CXCL10 induction in human pancreatic Death Differ 2014; 21: 956-66 Valeri N, Braconi C, Gasparini P, Murgia C, Lampis Giner FC, Rodenas-Cuadrado P, Mallappa S, Clark cancer stroma influences lymphocytes recruitment A, Paulus-Hock V, Hart JR, Ueno L, Grivennikov SI, S, Thomas H, Jeffery R, Poulsom R, Rodriguez- and correlates with poor survival. Oncotarget 2014 ; Ragusa S, Cheng J, Ivanov KI, Zangger N, Ceteci F, Lovat F, Paone A, Cascione L, Sumani KM, Justo M, Novelli M, Chetty R, Silver A, Sansom OJ, 5: 11064-80 Bernier-Latmani J, Milatos S, Joseph JM, Tercier S, Veronese A, Fabbri M, Carasi S, Alder H, Lanza G, Greten FR, Wang LM, East JE, Tomlinson I, Bouzourene H, Bosman FT, Letovanec I, Marra G, Gafa R, Moyer MP, Ridgway RA, Cordero J, Nuovo Leedham SJ. Aberrant epithelial GREM1 expression Meniel V, Megges M, Young MA, Cole A, Sansom Gonzalez M, Cammareri P, Sansom OJ, Delorenzi GJ, Frankel WL, Rugge M, Fassan M, Groden J, Vogt initiates colonic tumorigenesis from cells outside OJ, Clarke AR. Apc and p53 interaction in DNA M, Petrova TV. PROX1 promotes metabolic PK, Karin M, Sansom OJ, Croce CM. MicroRNA- the stem cell niche. Nat Med 2015; 21: 62-70. doi: damage and genomic instability in hepatocytes. adaptation and fuels outgrowth of Wnt(high) 135b promotes cancer progression by acting as a 10.1038/nm.3750. Epub 2014 Dec 1 Oncogene 2014 Oct 27. doi: 10.1038/onc.2014.342. metastatic colon cancer cells. Cell Rep 2014; 8: downstream effector of oncogenic pathways in [Epub ahead of print] 1957-73 colon cancer. Cancer Cell 2014; 25: 469-83 Faller WJ, Jackson TJ, Knight JR, Ridgway RA, Jamieson T, Karim SA, Jones C, Radulescu S, Huels Morran DC, Wu J, Jamieson NB, Mrowinska A, Rai TS, Cole JJ, Nelson DM, Dikovskaya D, Faller W, Walz S, Lorenzin F, Morton J, Wiese KE, von Eyss B, DJ, Myant KB, Dudek KM, Casey HA, Scopelliti A, Kalna G, Karim SA, Au AY, Scarlett CJ, Chang DK, Vizioli MG, Hewitt RN, Anannya O, McBryan T, Herold S, Rycak L, Dumay-Odelot H, Karim S, Cordero JB, Vidal M, Pende M, Ryazanov AG, Pajak MZ, Oien KA, McKay CJ, Carter CR, Gillen G, Manoharan I, van Tuyn J, Morrice N, Pchelintsev Bartkuhn M, Roels F, Wustefeld T, Fischer M, Sonenberg N, Meyuhas O, Hall MN, Bushell M, Champion S, Pimlott SL, Anderson KI, Evans TR, NA, Ivanov A, Brock C, Drotar M, Nixon C, Clark W, Teichmann M, Zender L, Wei CL, Sansom O, Wolf E, Willis AE, Sansom OJ. mTORC1-mediated Grimmond SM, Biankin AV, Sansom OJ, Morton JP. Sansom OJ, Anderson KI, King A, Blyth K, Adams Eilers M. Activation and repression by oncogenic translational elongation limits intestinal tumour Targeting mTOR dependency in pancreatic cancer. PD. HIRA orchestrates a dynamic chromatin MYC shape tumour-specific gene expression Gut 2014; 63: 1481-9 landscape in senescence and is required for profiles.Nature 2014; 511: 483-7 suppression of neoplasia. Genes Dev 2014; 28: 2712-25

84 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 85 Other Publications Other Publications Hock AK, Vigneron A, Vousden KH. Ubiquitin Peter D. Adams (page 62) Sansom O, Dhillon P. Translating the molecular Yu X, Narayanan S, Vazquez A, Carpizo DR. Small specific peptidase 42 (USP42) functions to Epigenetics of Cancer and Ageing hallmarks of colorectal cancer to patient therapies: molecule compounds targeting the p53 pathway: deubiquitylate histones and regulate transcriptional an interview with Owen Sansom. Dis Model Mech are we finally making progress?Apoptosis 2014; 19: activity. J Biol Chem 2014; 289: 34862-70 2014; 7: 937-40 1055-68 Primary Research Papers Haas NB, Quirt I, Hotte S, McWhirter E, Polintan R, Labuschagne CF, van den Broek NJ, Mackay GM, Litwin S, Adams PD, McBryan T, Wang L, Martin LP, Vousden KH, Maddocks OD. Serine, but not vonMehren M, Alpaugh RK, Zweibel J, Oza A. glycine, supports one-carbon metabolism and Emma Shanks (page 57) Marcos Vidal (page 42) Phase II trial of vorinostat in advanced melanoma. proliferation of cancer cells. Cell Rep 2014; 7: RNAi Screening Drosophila Approaches to Cancer 2014; 32: 526-34 1248-58 Invest New Drugs

Primary Research Papers Primary Research Papers Hofmann JW, McBryan T, Adams PD, Sedivy JM. Paulus-Hock V, Cheung EC, Roxburgh P, Vousden Jerby-Arnon L, Pfetzer N, Waldman YY, McGarry L, Cordero JB, Ridgway RA, Valeri N, Nixon C, Frame The effects of aging on the expression of Wnt KH, Hock AK. iRFP is a real time marker for James D, Shanks E, Seashore-Ludlow B, Weinstock MC, Muller WJ, Vidal M, Sansom OJ. c-Src drives pathway genes in mouse tissues. 2014; transformation based assays in high content Age (Dordr) A, Geiger T, Clemons PA, Gottlieb E, Ruppin E. intestinal regeneration and transformation. 36: 9618 EMBO J screening. PLoS One 2014; 9: e98399 Predicting cancer-specific vulnerability via data- 2014; 33: 1474-91 driven detection of synthetic lethality. 2014; Lund K, Cole J, VanderKraats ND, McBryan T, Cell Phesse TJ, Myant KB, Cole AM, Ridgway RA, 158: 1199-209 Faller WJ, Jackson TJ, Knight JR, Ridgway RA, Pchelintsev NA, Clark W, Copland M, Edwards JR, Pearson H, Muncan V, van den Brink GR, Vousden Jamieson T, Karim SA, Jones C, Radulescu S, Huels Adams PD. DNMT inhibitors reverse a specific KH, Sears R, Vassilev LT, Clarke AR, Sansom OJ. Mardilovich K, Gabrielsen M, McGarry L, Orange DJ, Myant KB, Dudek KM, Casey HA, Scopelliti A, signature of aberrant promoter DNA methylation Endogenous c-Myc is essential for p53-induced C, Patel R, Shanks E, Edwards J, Olson MF. Elevated Cordero JB, Vidal M, Pende M, Ryazanov AG, and associated gene silencing in Acute Myeloid apoptosis in response to DNA damage in vivo. Cell LIM kinase 1 in non-metastatic prostate cancer Sonenberg N, Meyuhas O, Hall MN, Bushell M, Leukemia. 2014; 15: 406 Death Differ 2014; 21: 956-66 Genome Biol reflects its role in facilitating androgen receptor Willis AE, Sansom OJ. mTORC1-mediated nuclear translocation. 2015; 14: translational elongation limits intestinal tumour Nelson DM, McBryan T, Jeyapalan JC, Sedivy JM, Mol Cancer Ther Stindt MH, Muller PA, Ludwig RL, Kehrloesser S, 246-58. doi: 10.1158/1535-7163.MCT-14-0447. initiation and growth. 2015; 517: 497-500. Adams PD. A comparison of oncogene-induced Nature Dotsch V, Vousden KH. Functional interplay Epub 2014 Oct 24 doi: 10.1038/nature13896. Epub 2014 Nov 5 senescence and replicative senescence: between MDM2, p63/p73 and mutant p53. implications for tumor suppression and aging. Oncogene 2014 Nov 24. doi: 10.1038/onc.2014.359. Age Other Publications Macagno JP, Diaz Vera J, Yu Y, MacPherson I, 2014; 36: 9637 [Epub ahead of print] (Dordr) Shanks EJ. Strategic siRNA screening approaches to Sandilands E, Palmer R, Norman JC, Frame M, target cancer at the Cancer Research UK Beatson Vidal M. FAK acts as a suppressor of RTK-MAP Rai TS, Cole JJ, Nelson DM, Dikovskaya D, Faller W, Other Publications Institute. 2014; kinase signalling in Drosophila melanogaster Vizioli MG, Hewitt RN, Anannya O, McBryan T, Comb Chem High Throughput Screen Lee P, Vousden KH, Cheung EC. TIGAR, TIGAR, 17: 328-32 epithelia and human cancer cells. 2014; Manoharan I, van Tuyn J, Morrice N, Pchelintsev PLoS Genet burning bright. Cancer Metab 2014; 2: 1 10: e1004262 NA, Ivanov A, Brock C, Drotar M, Nixon C, Clark W, Sansom OJ, Anderson KI, King A, Blyth K, Adams Maddocks OD, Labuschagne CF, Vousden KH. Parisi F, Stefanatos RK, Strathdee K, Yu Y, Vidal M. PD. HIRA orchestrates a dynamic chromatin Alexei Vazquez (page 20) Localization of NADPH production: a wheel within Transformed epithelia trigger non-tissue- landscape in senescence and is required for Mathematical Models of Metabolism a wheel. Mol Cell 2014; 55: 158-60 autonomous tumor suppressor response by suppression of neoplasia. Genes Dev 2014; 28: adipocytes via activation of Toll and Eiger/TNF 2712-25 Primary Research Papers Muller PA, Vousden KH. Mutant p53 in cancer: new signaling. Cell Rep 2014; 6: 855-67 Cicchini M, Chakrabarti R, Kongara S, Price S, functions and therapeutic opportunities. Cancer Nahar R, Lozy F, Zhong H, Vazquez A, Kang Y, Cell 2014; 25: 304-17 Scopelliti A, Cordero JB, Diao F, Strathdee K, White Karantza V. Autophagy regulator BECN1 suppresses BH, Sansom OJ, Vidal M. Local control of intestinal mammary tumorigenesis driven by WNT1 stem cell homeostasis by enteroendocrine cells in activation and following parity. Autophagy 2014; 10: the adult Drosophila midgut. Curr Biol 2014; 24: Sara Zanivan (page 44) 2036-52 1199-211 Vascular Proteomics

Lozy F, Cai-McRae X, Teplova I, Price S, Reddy A, Primary Research Papers Bhanot G, Ganesan S, Vazquez A, Karantza V. Guo Z, Neilson LJ, Zhong H, Murray PS, Zanivan S, ERBB2 overexpression suppresses stress-induced Karen Vousden (page 22) Zaidel-Bar R. E-cadherin interactome complexity autophagy and renders ERBB2-induced mammary Tumour Suppression and robustness resolved by quantitative tumorigenesis independent of monoallelic Becn1 proteomics. Science Signaling 2014; 7: rs7 loss. Autophagy 2014; 10: 662-76 Primary Research Papers D’Alessandro A, Amelio I, Berkers CR, Antonov A, van den Biggelaar M, Hernandez-Fernaud JR, van Warita K, Warita T, Beckwita CH, Schurdak ME, Vousden KH, Melino G, Zolla L. Metabolic effect of den Eshof BL, Neilson LJ, Meijer AB, Mertens K, Vazquez A, Wells A, and Oltvai ZN. TAp63alpha: enhanced glycolysis and pentose Zanivan S. Quantitative phosphoproteomics unveils Statin-induced mevalonate pathway inhibition phosphate pathway, resulting in increased temporal dynamics of thrombin signaling in human attenuates the growth of mesenchymal-like cancer antioxidant defense. 2014; 5: 7722-33 Oncotarget endothelial cells. Blood 2014; 123: e22-36 cells that lack functional E-cadherin mediated cell cohesion. 2014; 4: 7593 Haller M, Hock AK, Giampazolias E, Oberst A, Sci Rep Other Publications Green DR, Debnath J, Ryan KM, Vousden KH, Tait Reid S, Hernandez-Fernaud JR, Zanivan S. In vivo SWG. Ubiquitination and proteasomal degradation quantitative proteomics for the study of of ATG12 regulates its pro-apoptotic activity, oncometabolism. Methods Enzymol 2014; 543: 2014; 10: 2269-78 Autophagy 235-59

86 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 87 Soni S, Pchelintsev N, Adams PD, Bieker JJ. Other Publications Todt F, Cakir Z, Reichenbach F, Emschermann F, The winner of this year’s Award was Cassie Clarke Transcription factor EKLF (KLF1) recruitment of the Kamphorst JJ, Murphy DJ. The Beatson Lauterwasser J, Kaiser A, Ichim G, Tait SWG, Frank from Jim Norman’s group. Cassie is studying the histone chaperone HIRA is essential for beta-globin International Cancer Conference: Powering the S, Langer HF, Edlich F. Differential retrotranslocation role of RNA polymerase III in extracellular matrix gene expression. Proc Natl Acad Sci U S A 2014; 111: Cancer Machine. Cancer Metab 2014; 2: 25 of mitochondrial Bax and Bak. EMBO J 2015; 34: remodelling and cell migration. 13337-42 67-80. doi: 10.15252/embj.201488806. Epub 2014 Nov 5 Other publications Daniel J. Murphy (page 68) Theses Adams P, Antebi A, Cuervo AM, Kennedy B, Oncogene-Induced Vulnerabilities Other Publications Chaneton, Barbara (2014) Targeting cancer cell Sedivy J. Aging Cell: open access to all. Aging Cell Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson metabolism as a therapeutic strategy [PhD thesis, 2014; 13: 1 SA, Abrams JM, Adam D, Alnemri ES, Altucci L, Primary Research Papers University of Glasgow, Beatson Institute] Andrews D, Annicchiarico-Petruzzelli M, Muthalagu N, Junttila MR, Wiese KE, Wolf E, Baehrecke EH, Bazan NG, Bertrand MJ, Bianchi K, Morton J, Bauer B, Evan GI, Eilers M, Murphy DJ. Cuesta Garcia, Nerea (2013) P53 loss and KRAS Blagosklonny MV, Blomgren K, Borner C, Bredesen David Bryant (page 64) BIM is the primary mediator of MYC-induced mutation in an invasive murine model of colorectal DE, Brenner C, Campanella M, Candi E, Cecconi F, Molecular Control of Epithelial Polarity apoptosis in multiple solid tissues. 2014; 8: cancer [MRes thesis, University of Glasgow, Cell Rep Chan FK, Chandel NS, Cheng EH, Chipuk JE, 1347-53 Beatson Institute] Cidlowski JA, Ciechanover A, Dawson TM, Dawson Primary Research Papers VL, De Laurenzi V, De Maria R, Debatin KM, Di Other Publications Davidson, Andrew (2014) The role of WASP family Bryant DM, Roignot J, Datta A, Overeem AW, Kim Daniele N, Dixit VM, Dynlacht BD, El-Deiry WS, Kamphorst JJ, Murphy DJ. The Beatson members in Dictyostelium discoideum cell M, Yu W, Peng X, Eastburn DJ, Ewald AJ, Werb Z, Fimia GM, Flavell RA, Fulda S, Garrido C, Gougeon International Cancer Conference: Powering the migration [PhD thesis, University of Glasgow, Mostov KE. A molecular switch for the orientation ML, Green DR, Gronemeyer H, Hajnoczky G, Cancer Machine. 2014; 2: 25 Beatson Institute] of epithelial cell polarization. Dev Cell 2014; 31: Cancer Metab Hardwick JM, Hengartner MO, Ichijo H, Joseph B, 171-87 Jost PJ, Kaufmann T, Kepp O, Klionsky DJ, Knight Foth, Mona (2014) The role of FGFR3 mutation in RA, Kumar S, Lemasters JJ, Levine B, Linkermann tumour initiation, progression and invasion of Tran CS, Eran Y, Ruch TR, Bryant DM, Datta A, (page 70) Stephen Tait A, Lipton SA, Lockshin RA, Lopez-Otin C, Lugli E, urothelial cell carcinoma in mice [PhD thesis, Brakeman P, Kierbel A, Wittmann T, Metzger RJ, Mitochondria and Cell Death Madeo F, Malorni W, Marine JC, Martin SJ, University of Glasgow] Mostov KE, Engel JN. Host cell polarity proteins Martinou JC, Medema JP, Meier P, Melino S, participate in innate immunity to Pseudomonas Primary Research Papers Mizushima N, Moll U, Munoz-Pinedo C, Nunez G, Leach, Joshua (2014) Characterising the tumour aeruginosa infection. Cell Host Microbe 2014; 15: Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM. Oberst A, Panaretakis T, Penninger JM, Peter ME, microenvironment in pancreatic cancer and the 636-43 Using enhanced-mitophagy to measure Piacentini M, Pinton P, Prehn JH, Puthalakath H, changes elicited by targeted therapies [MRes thesis, autophagic flux.Methods 2015; 75: 105-11. doi: Rabinovich GA, Ravichandran KS, Rizzuto R, University of Glasgow, Beatson Institute] 10.1016/j.ymeth.2014.11.014. Epub 2014 Dec 9 Rodrigues CM, Rubinsztein DC, Rudel T, Shi Y, Jurre Kamphorst (page 66) Simon HU, Stockwell BR, Szabadkai G, Tait SW, Liu, Emma Yu (2014) Characterisation of the role of Cancer Metabolomics Haller M, Hock AK, Giampazolias E, Oberst A, Tang HL, Tavernarakis N, Tsujimoto Y, Vanden autophagy in DNA damage repair [PhD thesis, Green DR, Debnath J, Ryan KM, Vousden KH, Tait Berghe T, Vandenabeele P, Villunger A, Wagner EF, University of Glasgow, Beatson Institute] Primary Research Papers SWG. Ubiquitination and proteasomal degradation Walczak H, White E, Wood WG, Yuan J, Zakeri Z, Fan J, Ye J, Kamphorst JJ, Shlomi T, Thompson CB, of ATG12 regulates its pro-apoptotic activity, Zhivotovsky B, Melino G, Kroemer G. Manoharan, Indrani (2014) Identification and Rabinowitz JD. Quantitative flux analysis reveals Autophagy 2014; 10: 2269-78 Essential versus accessory aspects of cell death: characterization of novel histone modifications folate-dependent NADPH production. Nature recommendations of the NCCD 2015. Cell Death during cellular senescence [PhD thesis, University 2014; 510: 298-302 Orozco S, Yatim N, Werner MR, Tran H, Gunja SY, Differ 2015; 22: 58-73. doi: 10.1038/cdd.2014.137. of Glasgow, Institute of Cancer Sciences] Tait SW, Albert ML, Green DR, Oberst A. RIPK1 both Epub 2014 Sep 19 Kamphorst JJ, Chung MK, Fan J, Rabinowitz JD. positively and negatively regulates RIPK3 Rud-Majani, Zahra Erami (2014) Investigation of Quantitative analysis of acetyl-CoA production in oligomerization and necroptosis. Cell Death Differ Ichim G, Tait SW. E-cadherin dynamics in cancer cell adhesion and hypoxic cancer cells reveals substantial 2014; 21: 1511-21 Necroptosis: fifty shades of RIPKs. Mol Cell Oncol metastasis [PhD thesis, University of Glasgow, contribution from acetate. Cancer Metab 2015; 2. doi: 10.4161/23723548.2014.965638. Beatson Institute] 2014; 2: 23 Epub 2014 Nov 7 Steele, Colin (2014) Investigating the role of CXCR2 Lopez J, Tait SW. signalling in pancreatic inflammation and cancer Killing the Killer: PARC/CUL9 promotes cell survival [PhD thesis, University of Glasgow, Beatson by destroying cytochrome C. Sci Signal 2014; 7: Institute] pe17

Tait SW, Ichim G, Green DR. Die another way - non-apoptotic mechanisms of cell death. J Cell Sci 2014; 127: 2135-44

John Paul Career Award All third year PhD students at the Beatson are eligible for this Award, named after Dr John Paul, the founding Director of the Institute. Candidates prepare a progress report on their work and give a talk to staff and other students.

88 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE RESEARCH PUBLICATIONS 89 CONFERENCES AND WORKSHOPS

Cancer Research UK Beatson route to treatment of metastatic disease will also be Open Evening International Cancer Conference discussed (see www.beatson.gla.ac.uk/conf for Our opening evening, held in March during from studying cell death and cell migration to Powering the Cancer Machine more details and to register). National Science and Engineering Week, was again treating pancreatic cancer and brain tumours. 6 - 9 July 2014 very well attended. There were talks, given by These were followed a series of lab tours and Bute Hall, University of Glasgow In addition, we will be hosting a workshop Stephen Tait, Emma Woodham, Colin Steele and demos by our hardworking volunteers. Scientific Committee: Jim Norman, Eyal Gottlieb, entitled ‘Next Generation Cancer Biology’ in Anthony Chalmers, covering topics that ranged Daniel Murphy, Karen Vousden September, which will focus on the growing role of systems analysis and data models in modern Poster for 2015 bioscience research. This year’s conference focused on cancer conference metabolism, an increasing important area of CANCER RESEARCH UK research for the Beatson as well as for the wider BEATSON INTERNATIONAL CONFERENCE Co-sponsor WORLDWIDE CANCER RESEARCH (formerly known as AICR) cancer research community. The participants heard Fluorescence Lifetime and FRET from a range of excellent speakers including David Microscopy Workshop Control of Cell Polarity and Movement in Cancer Sabatini (Cambridge, MA) who gave a 3 & 4 September 2014 Sunday 5 July - Wednesday 8 July 2015 CRUK Beatson Institute comprehensive overview of what is known about Speakers and Sessions: the regulation and functions of the mTOR pathway Keynote Address: in the Colin Thomson Memorial Keynote Lecture This meeting, sponsored by Nikon UK and the Royal Ian Macara (US) on the first evening. This and the preceding Microscopical Society, consisted of presentations Opening Session: Margaret Frame (UK) opening session were sponsored by Worldwide by invited speaker Simon Ameer-Beg (Kings College Xin Lu (UK) London), head of the Beatson Advanced Imaging Cancer Research (WCR, formerly known as AICR). Self-Generated Gradients/ Resource Kurt Anderson, Nikon Microscopy UK, Mechanical Forces: Selected short talks were also given by Julianna Darren Gilmour (DE) Blagih, Maria José Bueno, Brooke Emerling, LaVision Biotec and Lambert Instruments along Rob Insall (UK), Johan de Rooij (NL) Fernando Martin-Belmonte (ES), Alpha Yap (AU) Sarah-Maria Fendt, Maria Georgiadou, Hannah with a series of practical hands-on workshops and demonstrations on the use of FLIM-FRET. Cell Intrinsic Polarity/ Johnston, Patricia Sancho, Darren Saunders, Cell Migrations: Buzz Baum (UK) Saverio Tardito, Caroline Treins (sponsored by WCR) Alexander Bershadsky (SG/IL) and Hans-Guido Wendel (sponsored by Boehringer Dave Bryant (UK) Angeliki Malliri (UK) Ingelheim). Caroline Treins (Paris) won the Portland Postdoc Meeting Jacco van Rheenan (NL) Visualizing Cancer: Microscopy and Beyond Press sponsored prize for her talk describing the Signalling Pathways to Generation/ identification of novel molecular targets of S6Ks, 12 September 2014 Loss of Epithelial Polarity: CRUK Beatson Institute David Bilder (US), Andrew Ewald (US) while Katarzyna Grzes (Dundee) was awarded the Barry Thompson (UK) Marcos Vidal (UK), Jeff Wrana (CA) AMSBIO-Trevigen sponsored poster prize for her Organisers: Amelie Juin (Beatson), Martin Lee (Edinburgh), Daniel Soong (Edinburgh), Haoran Membrane Trafficking, work investigating metabolic regulation in PTEN Cell Migration and Polarity: Tang (Manchester), Ben Tyrrell (Beatson) Yohanns Bellaiche (FR) null T lymphoma cells. The meeting was Enrique Rodriguez-Boulan (US) generously co-sponsored by Cancer Research UK Johanna Ivaska (FI) In September, our postdocs, along with a number Jim Norman (UK) and Worldwide Cancer Research. A report of the Roberto Weigert (US) from Edinburgh and Manchester, hosted a one-day meeting was published in Cancer & Metabolism Short talks will be granted to the authors of outstanding abstracts. Some financial assistance will be available to the presenters of these talks through sponsorship from Worldwide Cancer Research (Kamphorst & Murphy. Cancer Metab 2014; 2: 25). meeting at the Institute with funding from the American Society for Cell Biology. This included Website, on-line registration, payment and abstract submission instructions: http://www.beatson.gla.ac.uk/conf For additional information please contact: Conference Administrator, Beatson Institute for Cancer Research, talks and poster presentations by postdocs and Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK The 2015 meeting will highlight recent exciting Tel: +44(0) 141 330 3953 Fax: +44(0) 141 942 6521 • Email: [email protected] Deadline for registration, payment and abstract submission: Monday 11 May 2015 research into the molecular and cellular events that students as well as keynote lectures by Jeff Pollard contribute to loss of epithelial polarity during (University of Edinburgh) and Erik Sahai (London carcinogenesis, and how cancer cells acquire Research Institute). Following the success of this different types of polarity that enable them to meeting, our postdocs hope to follow up with migrate and invade. The development of agents to another meeting next year. target cells with aberrant polarity as a potential

90 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE CONFERENCES AND WORKSHOPS 91 SEMINARS AT THE BEATSON INSTITUTE

The following seminars were held at the CRUK Beatson July Institute during 2014. Helfrid Hochegger, Genome Damage and Stability Centre, University of Sussex Jason Carroll, Cancer Research UK Cambridge January April Institute Brian Stramer, Randall Division of Cell and Rob Liskamp, School of Chemistry, University of Molecular Biophysics, King’s College London Glasgow Robert Kay, MRC Laboratory of Molecular Biology, Thomas Tuetling, University of Bonn, Germany September University of Cambridge Tor Henrik Semb, The Danish Center for Stem Cell Andrea Musacchio, Max Planck Institute of Martin Humphries, Faculty of Life Sciences, Research, Denmark Molecular Physiology, Dortmund, Germany University of Manchester Bruno Galy, European Molecular Biology Markus Rehm, Royal College of Surgeons, Ireland Lena Claesson-Welsh, Department of Laboratory, Heidelberg, Germany Immunology, Genetics and Pathology, Uppsala Alberto Signore, Nuclear Medicine Unit, ‘Sapienza’ University, Sweden University of Rome, Italy October Thomas Radimerski, Novartis, Switzerland Michael Overholtzer, Memorial Sloan Kettering Cancer Center, New York, NY, USA May Mathew Coleman, School of Cancer Sciences, February Eytan Ruppin, School of Computer Sciences & University of Birmingham Nitzan Rosenfeld, Cancer Research UK School of Medicine, Tel Aviv University, Israel Antony Braithwaite, Otago University, Cambridge Institute Joel Leverson, Abbvie Pharmaceuticals, Chicago, New Zealand IL, USA Bader Al-Anzi, California Institute of Technology, Pasadena, CA, USA March Oliver Bischof, CNRS-CR1, Institut Pasteur, Paris Peter Vandenabeele, Flanders Institute for Darren Gilmour, Cell Biology and Biophysics Unit Michael Lisanti, Breakthrough Breast Cancer Biotechnology, Belgium at EMBL, Heidelberg, Germany Research Unit, University of Manchester Robert Gillies, Chair, Cancer Imaging and Metabolism, Moffitt Cancer Centre, Tampa, FL, USA June November Andrew Renault, School of Life Sciences, Vassiliki Kostourou, Biomedical Sciences Research Christopher Kemp, Fred Hutchinson Cancer University of Nottingham Centre ‘Al. Fleming’ Vari, Attiki, Greece Research Center, Seattle, WA, USA Martin Schwartz, R.W. Berliner Professor of Lorraine O’Reilly, The Walter and Eliza Hall Institute Alex Gould, MRC National Institute for Medical Medicine and Cell Biology, Yale School of Medicine, (WEHI), Melbourne, Australia Research, London New Haven, CT, USA Andreas Strasser, Walter and Eliza Hall Institute, James Bear, Department of Cell Biology and Melbourne, Australia Physiology, Lineberger Comprehensive Cancer Center, NC, USA Federica Maione, Institute for Cancer Research, Candiolo, Turin, Italy

92 SCIENTIFIC REPORT 2014 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 relationships with Cancer Research UK, that the Beatson Institute complies with all relevant the University of Glasgow and many other our mission to support cancer research of the highest standard. Our regulations and adopts best practice in financial, organisations with which our scientists aim is to continue to attract enthusiastic scientists and clinicians early in personnel and corporate governance matters. Our have contact. Finance & Human Resources team has been set up their careers to work with our established staff and to draw on their to do this, providing the Institute’s Board of Scientific Administration experience but also to spark new ideas in a stimulating research Governors with necessary financial and legal Jackie Beesley, Catherine Winchester information, ensuring that funding bodies’ environment. As well as learning a very wide range of practical and administrative requirements are met and that The Scientific Administration team supports technical skills our junior researchers participate in all intellectual funding for the Institute’s research activities is scientists at the Beatson Institute by taking minutes properly managed. at a range of scientific and administrative meetings, activities and present and discuss their own work at internal seminars editing publications such as the Scientific Report, and external meetings. We provide support and facilities of the highest The Beatson Institute also needs to coordinate with maintaining an up-to-date website, administering the University of Glasgow’s central administration the graduate student training programme, and standards and scientific interactions are encouraged by our over such matters as the administration of grants, providing reports and answering queries for both international conference, workshops and seminars and by funding payment of suppliers and staffing. Our Finance & internal and external audiences, including Cancer Human Resources and Secretarial teams provide Research UK. The team also assists researchers in participation in external meetings. that vital link. identifying and applying for external grant funding and has a role in providing oversight , training and advice in all aspects of research integrity. PhD Studentships administrative matters required in relation to the Finance & Human Resources The purpose of our clinician/graduate training University or funding body. The advisor gives Peter Winckles, Caroline Preacher programme is to give students and clinical fellows additional guidance by providing independent Jacqui Clare, Karen Connor, Cancer Research Technology starting in research an opportunity to work in advice on any matters concerning the studentship Nicki Kolliatsas, Elaine Marshall, Frank Maria Lopalco state-of-the-art laboratories. This enables them to and by attempting to resolve any problems that McGeoghegan, Gary Niven, Lynn Wilson, assess and develop their research talents to the full, may arise. Patricia Wylie Cancer Research Technology (CRT) is an to decide whether a research career suits them and oncology-focused technology transfer and to use their period of graduate study as a Postdoctoral Research Scientists and Fellows The Finance & Human Resources team is development company wholly owned by Cancer springboard for their future career path. Our We see postdocs as pillars of the research and responsible for all accounting and personnel Research UK with 130 employees based primarily four-year studentships are designed to give intellectual activities of their own groups and of the management issues including banking, payments, in London and Cambridge. Since a substantial graduates who show a strong aptitude for research Beatson Institute as a whole. The training grants management, budgeting, pay amount of the funding for the Beatson Institute the opportunity to complete substantial research programme is designed to promote the administration, staff policies and procedures, and comes from Cancer Research UK, CRT manages projects resulting in very good publications. All development of outstanding and dedicated junior reporting financial information for funders, all intellectual property-related matters on behalf students receive training in safe working practices, scientists and we hope that by the end of their managers and the Board of Directors. of the Institute and the charity. To facilitate this, writing project reports and other transferable skills. tenure they will be ready to compete for an there is a CRT Business Manager based full-time at Training also involves learning to be an independent independent position. Postdocs are initially the Institute. 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 Sheila McNeill (PA to Professor Vousden), seminars and actively contributing to scientific scientists may be given additional support and Catriona Entwistle, Rebecca Gebbie, Barbara Laing, discussions. Students are also given the opportunity responsibility – such as more technical help or 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 required for the project and dealing with any useful links.

94 SCIENTIFIC REPORT 2014 CANCER RESEARCH UK BEATSON INSTITUTE ADMINISTRATION 95 THANKS FOR SUPPORTING US

The work of our various research groups would barely proceed without Beatson Associates · Irene Craig · J Allan Denholm, in memory of the Late Mr the substantial grant funding provided by Cancer Research UK to the Peter Adams Jim Colraine Beatson Institute and the University of Glasgow, now amounting to BBSRC, Medical Research Council, NIH with · Edrington National Institute on Aging & National Cancer · Fisher Scientific UK over £18 million per annum combined. We are also indebted to a Institute (USA) · May Gow, Rozelle House, Ayr number of other organisations that provide funding to our scientists, · Avril Haddow Daniel Murphy · B Herbert, in memory of Miss Elizabeth usually supporting projects in a particular sphere of special interest, or British Lung Foundation, European Community, (Bunty) Cameron supporting the careers of talented junior scientists, enabling them to Merck Sharp & Dohme, Worldwide Cancer · Hodder & Stoughton Ltd (Hachette), re Royalties Research · Hyndland Parish Church pursue their research interests within our laboratories. These · The James Inglis Trust organisations, whose funding we appreciate greatly, are listed below. Stephen Tait · Sameena Kelly, Glasgow Kelvin College ARC, BBSRC, EMBO, EU Marie Curie Actions, · David Kerr The additional funding provided by these organisations makes possible Royal Society · Kaitlin Kimmins, Glasgow Kelvin College much work that we otherwise could not be undertaking and has · Kings Park Parish Church, Ladies Badminton Club We do not purposefully solicit contributions to our · Janetta Helen Kinloch, in memory of become integral and indispensable to our operations. work directly from the general public – we see this Bunty Cameron as the role of the cancer charities such as those that · Lanarkshire Women’s Bowling Association feature above. We are, however, fortunate to be in · Agnes MacDonald Jim Norman Cancer Research UK Beatson Institute the minds of many local people and organisations · Marks & Clerk Breast Cancer Campaign) who give generously of their time and effort to raise · Legacy from the Estate of the Late Mr & Mrs Kurt Anderson funds for good causes. We are also, more Finlay McCulloch Bruker, Technology Strategy Board, Novartis Michael Olson poignantly, in the minds of those who are suffering · Helen McCulloch Breast Cancer Campaign, Medical Research cancer, or who have lost loved ones to this disease. · Christina McDougall, in memory of her Council, Worldwide Cancer Research Karen Blyth To those who give time and effort to raise funds on husband John Breast Cancer Campaign, Royal Society our behalf and to those who thoughtfully regard · Liz McGinniss Kevin Ryan us as suitable beneficiaries of their generosity, · Fiona McNeill and family Martin Drysdale Astellas Pharma Inc, EMBO, Worldwide thank you. · McQuarrie, in memory of their daughter Rhoda Medical Research Council Cancer Research · Legacy from the Estate of the Late Mrs Jean Merry · Aggreko UK Ltd · Legacy from the Estate of the Late Mrs Jeff Evans Owen Sansom · Balfour & Manson LLP Agnes Middleton Scottish Executive - Chief Scientist Office, Bioven, European Community, Institute of Cancer · S3 and S5 pupils of Bellahouston Academy · B Millar Medical Research Council, Pancreatic Cancer Research, Janssen Pharmaceutica NV, Medical · Biogenes GmbH · Jim Millar Research Fund Research Council, Novartis, Royal Commission for Bioline Reagents · Legacy from the Estate of the Late Mrs Helen S the Exhibition of 1851, Wellcome Trust, Worldwide · · Margaret G Brown M Mitchell Eyal Gottlieb Cancer Research · Buchanan Castle Golf Club · MOD Police, RNAD Coulport AIRC (Italian Association for Cancer Research), · Brian Burns · Mosshead Primary School FEBS, Janssen Pharmaceutica NV, Metabomed Douglas Strathdee Cambridge Bioscience Ltd · North View Housing Association, Castlemilk Barth Syndrome Foundation · · Elizabeth M Campbell, in memory of Mr Family Day Hing Leung Alasdair Campbell · Anne O’Hare, Hillpark Bowling Club Academy of Medical Sciences, Medical Research Karen Vousden · Jane Carmichael, in memory of Miss · Dr & Mrs J D Olav Kerr’s Charitable Trust Council, Prostate Cancer Charity Astex, European Community, NHS Greater Elizabeth (Bunty) Cameron · Sarah Percy and Irene Kennedy Glasgow & Clyde Health Board Endowment Fund, · Cathcart Castle Golf Club Ladies Section · The Worthy Matron, Worthy Patron, Officebearers Laura Machesky West of Scotland Women’s Bowling Association, · Citywire and Members, Order Of The Eastern Star - Lily of Danish Council, Medical Research Council Worldwide Cancer Research · Clyde Travel the Valley · Legacy from the Estate of the Late Mrs · Mabel M Reid, in memory of her friend Sarah Colman Bunty Cameron · Ann P Coulson, in memory of Bunty Cameron

96 SCIENTIFIC REPORT 2014 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 and accountability. On a day-to-day basis, many of these responsibilities are delegated to the Institute’s Management Team.

Prof Nic Jones (Chair) Chief Scientist, Cancer Research UK

Mr Craig Anderson Senior Partner, KPMG

Mr Kirk Murdoch Laraine Kernahan visited Clyde Travel, Hillington to be presented with a cheque for over £5000 Chairman, Pinsent Masons, Scotland & Northern Ireland · In memory of Andy Robertson, from family · VWR and friends · J Walker Dr Iain Foulkes · Sarstedt Ltd · West of Scotland Women’s Bowling Association Executive Director, Strategy and Research Funding, · Scullion · Frank Wilson Cancer Research UK · Eileen Smillie · Legacy from the Estate of the Late Mr William · St Andrew of Glasgow Royal Arch Chapter No.69 Cluggie Wilson Mr Ian Kenyon · The Staypar Charitable Trust, in memory of the Chief Financial Officer, Cancer Research UK Late Gail Wylie · St Isidore’s Catholic Church Company Secretary · Members, friends and family of St Rollox Mr Peter Winckles Bowling Club Cancer Research UK Beatson Institute · St Vincent Bowling Club · George Taggart, on behalf of Mr & Mrs Ian · · McDonald’s Golden Wedding Anniversary · John Teevan, in memory of their late mother · Thermo Fisher · Thornhill Gardening Society · Vivienne Triseliotis, in memory of Bunty Cameron · Peter Vardy Foundation

98 SCIENTIFIC REPORT 2014 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 © 2014 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 2014 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