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Research involving animals

Research involving animals has contributed to many of the medical advancesin the detection and treatment of cancer that we now take for granted. These include MRI and CT scanning to detect cancer, anaesthetics and other techniques used in surgery, as well as radiotherapy, chemotherapy, anti-cancer drugs such as tamoxifen, and also pain relief and antibiotics to prevent infection.
Most of our research does not involve animals. However some animal research remains essential if we are to understand, prevent and cure cancer. Where animal research is undertaken at the Institute, it involves primarily mice, and to a much smaller extent rats and fruit flies.
Research involving animals enables us to understand basic physiological processes and the mechanisms underlying cancer. Alternatives to the use of animals, such as studying cancer cells growing in the lab and using computers to predict cancer cell behaviour have greatly reduced the number of animals used  and we are committed to developing alternatives to continue reducing animal use. However, at the moment, these alternatives cannot fully mimic the complex interactions that take place in the body.  Research at the Institute involving animals is not undertaken lightly and, in line with the values of Cancer Research UKand UK law, research involving animals only takes place where there is no alternative.
The welfare of our animals, and promoting a culture of care towards them, is a top priority. As a research community we closely follow the guidance published by animal welfare bodies such as the RSPCA and LASA (Laboratory Animals in Science Association). We are committed to the principles of the 3Rs: Replacement (methods which avoid or replace the use of animals), Reduction (methods which minimise the number of animals used and increase data per animal), and Refinement (methods which minimise suffering and improve animal welfare).
Where animal studies are unavoidable, they are conducted in strict compliance with the UK Animals (Scientific Procedures) Act 1986 and the EU Directive 2010/63 under the associated licensing process. As part of this process, the potential scientific and medical benefits of the research are weighed up against the possible effects on the animals involved by our Animal Welfare and Ethics Review Body and the Home Office before any research project involving animals can proceed. We have a dedicated team who guide and train our scientists, and oversee the care and welfare of the animals. We work closely with a veterinary surgeon to ensure that the highest levels of both staff competency and animal welfare are being maintained.
Both the University of Cambridge and Cancer Research UK are signatories of the Concordat on Openness on Animal Research in the UK which sets out how organisations report the use of animals in scientific, medical and veterinary research in the UK.

Research Group Leaders
Research Groups
•    Balasubramanian, Shankar
Chemical Biology
Recent advances in understanding nucleic acid function have shown that alternative secondary structures and the chemical modification of nucleotide bases have key regulatory roles in diverse cellular processes, from transcription and translation to cell division and genome stability.
Genetic information is carried not only by the sequence of nucleic acids, but also their secondary structures and chemical modifications. For example, guanine-rich sequences can form stable four stranded structures called G-quadruplexes (G4s), while certain cytosine bases in DNA can become methylated. We hypothesise that such alternative structures, or chemical modifications, have critical functions in normal cells and cancer. By identifying where base modifications and G4 structures are located in the cancer cell genome, and through the application of synthetic small molecules that selectively target G4 structures, we aim to understand the oncogenic process and develop novel approaches for potential use in treatment and diagnosis of cancer. We are also exploring new strategies to target the DNA binding activity of FOXM1, a key cancer-related transcription factor involved in cell cycle control.
In guanine (G)-rich regions, G bases can adopt stable arrangements to form four-stranded G4 structures comprising stacked G-tetrads (Figure 1).
Figure 1. G4 formation mediated by Hoogsteen hydrogen bonding between four guanines and co-ordinated by a central metal cation (left).  Stacked G-tetrads in an intramolecular G4 (right) (see Balasubramanian et al, Nat Rev Drug Discovery 2011; 10: 261).
Sequences with potential to fold into a G4 structure are common in the human genome and many are found in cancer-related genes such as KIT, RAS and SRC. G4s are implicated in biological processes ranging from chromosome stability to the regulation of gene transcription, and we seek to understand their biological function and validity as drug targets.
We are identifying where in the genome G4s form and their regulation in cancer phenotypes. By synthesising small chemical probes and engineering antibodies to recognise G4 structures with high specificity and affinity, we have visualised G4 formation G4 in the nuclei of cancer cells (Figure 2).
Figure 2. The G4 stabilising ligand, pyridostatin, leads to DNA damage at oncogenes including SRC. SRC gene structure is shown below (black).  The sites of DNA damage, indicated by the γH2AX marker, before (Unt) and after pyridostatin treatment (1) are shown above.  Predicted G4 sequences (PQS) in the SRC gene are indicated in purple (see Rodriguez et al., Nature Chem Biol. 2012; 8: 301).
Using chromatin immunoprecipitation and next generation sequencing (ChIP-Seq), we have localised G4 structures genome-wide in human DNA and shown that expression of G4-containing genes is modulated by small molecule ligands. Indeed, our G4-binding small molecule, pyridostatin (PDS), imparts growth arrest of human cancer cells through the activation of a DNA damage response in which sites of DNA damage localise to several oncogenes, including SRC (Figure 2). PDS causes down-regulation of SRC expression and inhibition of SRC-mediated cellular motility. Furthermore, we have found that G4 DNA is a molecular target for synthetic lethality of cancer cells since PDS acts synergistically when DNA repair pathways are inhibited or mutated. This work provides a novel framework for defining functional drug-DNA interactions and their potential use in cancer therapies.
During cell division, chromosome ends (telomeres) are protected from damage by recruitment of a protective protein complex called shelterin. Telomeres can form stable G4 structures in vitro and we have now used our antibody probe to demonstrate their presence at telomeres and across the genome in human cancer chromosomes (Figure 3).
Figure 3. Detection of G4 structures (red) in the nuclei (circles) of breast cancer cells using an engineered structure-specific antibody before (A) and after (B) stabilisation with pyridostatin.  C) Detection of G4 structures (red) in breast cancer metaphase chromosomes (blue), the arrow indicates localisation to the telomere.
As 85% of primary tumours show increased expression of the telomere maintenance enzyme telomerase, targeting telomeres may lead to cancer cell death. We have found that PDS treatment releases shelterin from telomeres leading to DNA damage and cell death. Telomeres are also known to be actively transcribed into a G4-containing telomeric RNA called TERRA. While TERRA forms stable G4s in vitro, it is not known whether this holds true in vivo. We have now demonstrated that shelterin proteins bind to TERRA via the G4 structure, and we are aiming to understand how this influences telomere function.
Predicted G4 structures are common in RNA, and their position suggests they have key roles in RNA biology. We have shown in vitro that a conserved G4 motif in the NRAS oncogene 5’-UTR modulates protein translation, and can be targeted by small molecule ligands. Despite this, the functional relevance of G4 structures is not known in vivo, nor whether G4 structures normally form in RNA. Recently, using our antibody probes, we have provided evidence for the presence of RNA G4s in the cell cytoplasm. We have also used a ‘click-chemistry’ procedure to synthesise small molecules that are selective for RNA over DNA G4s and used these in cells to selectively stabilise cytoplasmic RNA G4s. We are now using such chemical biology tools, together with genome-wide approaches, to identify and map RNA G4s within the transcriptome and to understand their roles in RNA biology in cancer cells.
Epigenetics and Modified Bases
Chemical modifications to DNA bases can affect the activity of genes. These epigenetic marks switch genes on and off in and the modified base 5-methylcytosine (5mC) is well known as to regulate transcription. Three further modified bases, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine have been found in the mammalian genome and are thought to arise from 5mC through the action of hydroxylases such as the TET family. Evidence is emerging that cancer cells have altered epigenetic profiles, though the function of these modifications in normal biology and their role in disease is not fully understood. We are developing tools to locate and study the function of these modified bases in genomic technologies and have invented oxidative bisulfite sequencing to quantitatively sequence 5mC and 5hmC at single-base resolution (Figure 4) . We are now focusing on the identification, mapping and elucidation of their biological function in normal and disease cells.
Figure 4. Oxidative bisulfite sequencing of 5mC and 5hmC. By oxidising 5hmC to 5fC followed by bisulfite treatment, C and 5hmC are read as T while 5mC is read as C. Without oxidation mC and 5hmC are read as C following bisulfite treatment, thus 5mC can be determined by subtraction (see Booth et al., Nat Protoc. 2013; 8: 1841).
As cancer cells have abnormal gene expression profiles, transcription factors represent an attractive target for intervention. We are exploring if transcription factors can be targeted by small molecules. Historically, successful drug design for such proteins has been limited as their surfaces tend to be hydrophobic and unremarkable, providing few druggable regions. However, as direct DNA-protein contacts are made when DNA binding domains (DBDs) associate with DNA, small molecules that disrupt this interaction may be promising. We now have proof-of-concept of this concept for FOXM1, a master cell cycle regulator implicated in oncogenesis. We have shown that the natural product thiostrepton binds to the FOXM1 DBD causing the genome-wide dissociation of the transcription factor from DNA in breast cancer cells. We have also found that the estrogen receptor (ERα) and FOXM1 bind simultaneously at genomic sites and that FOXM1 regulates ERα transcriptional activity through the co-activator, CARM1. Furthermore, FOXM1 inhibition using thiostrepton has uncovered of a set of FOXM1-regulated genes that correlate with patient outcome in clinical breast cancers. This demonstrates the potential druggability of an important regulatory mechanism and paves the way for the design of more potent and selective agents that target FOXM1 and other transcription factors.

•    Bohndiek, Sarah
•    The VISION Laboratory, led by Dr Sarah Bohndiek operates jointly between the Department of Physics and the Cancer Research UK Cambridge Institute.
•    In the Physics laboratory, we develop and validate new imaging technologies. We currently focus on signal excitation in the visible and near infrared and aim to produce hyperspectral imaging methods with high sensitivity and specificity for clinical translation.
•    In the CRUK CI laboratory, we combine these new developments in molecular imaging with preclinical disease models to better understand cancer therapy response and drug resistance. In particular, we are interested in the role of oxygen, including the presence, absence and metabolism of oxygen in tumour cells and surrounding blood vessels.

•    Brenton, James
Functional Genomics of Ovarian Cancer
Building a molecular classifier—how many types of high grade serous ovarian cancer are there?
The key genomic features of high grade serous ovarian carcinoma (HGSOC) are ubiquitous TP53 mutation and profound genomic instability causing highly abnormal copy number (CN) and structural variation (SV). We have exploited these to develop potential molecular classifications of high-grade serous ovarian cancer (HGSOC).
We have optimised our low-cost tagged amplicon sequencing of TP53, resulting in more accurate mutation calling and variant allele fraction estimation for FFPE and cell free DNA. We have used these assays to validate the performance of p53 IHC as a predictor of TP53 mutation and establish sensitivity and specificity parameters. Our methods have direct clinical impact and have been incorporated as exploratory endpoints in clinical trials In addition, we are translating these assays and analysis pipelines into the clinic for use in women with HGSOC.
Recently, we have adopted a low-cost, low-coverage, whole-genome sequencing approach to interrogate the copy-number landscape of HGSOC in a clinical setting. We have developed a bioinformatic method that estimates tumour purity and ploidy from shallow WGS using TP53 mutant allele fraction in order to get absolute copy-number. The application of this to a cohort of 300 samples has revealed distinct copy-number signatures.
Looking further forward we plan to leverage the observation made by others that subtypes of HGSOC have immune related gene expression signatures - we will overlay matched H&E image analysis, quantifying immune cell infiltration, with our tagged amplicon and copy-number sequencing to attain a more comprehensive classification.
Unlocking the clinical utility of ctDNA
Circulating tumour DNA is a promising non-invasive biomarker that can provide highly specific genomic information in women with HGSOC.
We have demonstrated that ctDNA TP53 mutant allele fraction is correlated with tumour volume and that a decrease after cycle 1 is associated with longer time to progression. It appears that ctDNA is promising as a predictive and prognostic biomarker in a relapsed setting and we are now validating these findings in further cohorts from other clinical trials.
ctDNA has the potential to provide biomarkers of high clinical utility for cancer diagnosis and monitoring in HGSOC. We will transfer our optimized ctDNA assays into the clinic for implementation in wider clinical trials.
Functional analysis of cellular heterogeneity in HGSOC
In vitro and in vivo tumour models to study HGSOC are very limited. Our aim is to establish reliable pre-clinical model systems that can represent the complex genomic profiles and intratumoural heterogeneity of HGSOC so that these reagents can be used in functional screens and the discovery of therapeutics and predictive biomarkers.
We have carried out systematic culture experiments to develop 2D cell lines which show that primary HGSOC cells have a highly reproducible limited lifespan when grown as in vitro. We have optimized conditions to grow primary ascites cells in suspension and have tested with RNAseq the similarity between primary ascites spheroids and established cell lines grown in 3D using low attachment media. We have started to develop methods for organoids growth from primary cells and demonstrate improved survival compared to 2D methods.
We are developing established HGSOC xenografts for long term maintenance of HGSOC populations and have optimized methods for tumour disaggregation, freezing and transplantation. To investigate the cellular heterogeneity within HGSOC, we have used a combination of multi-parameter flow cytometry and in vitro and in vivo functional assays to interrogate the growth and differentiation properties of phenotypically distinct cell populations present within freshly isolated, non-cultured HGSOC tissues.
We have established proof-of-principle methods for characterizing and screening patient-derived ascites spheroids in high-throughput assays and for the establishment and functional interrogation of PDX xenograft models.

•    Brindle, Kevin
Molecular Imaging (MRI and MRS)
The aim of our laboratory is to develop imaging methods that can be used in the clinic to detect early tumour responses to treatment.
The primary focus of our work is early detection of treatment response with the aim of developing imaging methods that could be used in early phase clinical trials to get an indication of drug efficacy, and subsequently in the clinic to guide treatment in individual patients (Brindle, Nat Rev Cancer2008; 8: 1). We have also started to work on the more challenging problem of early detection and the development of imaging methods that potentially could be used for patient screening.
Imaging metabolism with hyperpolarised 13C-labelled cell substrates
MRI gives excellent images of soft tissues, such as tumours. The technique works by imaging the distribution and MR properties of tissue water protons, which are very abundant (60 –70 M in tissues). However, we can also use MR to detect metabolites in vivo. The problem is that these molecules are present at ~10,000× lower concentration than the protons in tissue water, which makes them hard to detect and almost impossible to image, except at very low resolution. We have been collaborating with GE Healthcare in the development of a technique, termed ‘hyperpolarisation’, that increases sensitivity in the MRI experiment by more than 10,000×. With this technique we inject a hyperpolarised 13C-labelled molecule and now have sufficient sensitivity to image its distribution in the body and the distribution of the metabolites produced from it.
Previously we had shown we can detect early response to chemotherapeutic drugs by monitoring decreased tumour utilization of one cell metabolite, pyruvate, and then detect subsequent cell death by watching the increased metabolism of another metabolite, fumarate. We are taking these forward in a clinical trial, which is due to start in 2014, to detect treatment response in lymphoma, glioma and breast cancer patients. A clinical device for hyperpolarising 13C-labelled pyruvate and fumarate was installed in the Department of Radiology in Addenbrooke’s Hospital earlier this year. Detecting treatment response through the decrease in hyperpolarised 13C label exchange between injected pyruvate and endogenous lactate is analogous to monitoring response through measurements of the decrease in 18fluorodeoxyglucose (18FDG) uptake using Positron Emission Tomography (PET), and indeed we have compared these two methods directly. However, both methods only probe three steps in the glycolytic pathway, which is responsible for the metabolism of glucose to lactate and which is often highly up-regulated in tumour cells. These three steps are vascular delivery, plasma membrane transport (via the glucose transporters) and hexokinase activity, in the case of 18FDG; and vascular delivery, plasma membrane transport (via the monocarboxylate transporters) and lactate dehydrogenase activity, in the case of hyperpolarized [1-13C]pyruvate. Both the enzymes and the transporters are up-regulated in tumour cells. We have shown that by using a spin echo experiment that we can monitor the extravasation and tumour cell uptake of hyperpolarised [1-13C]lactate and pyruvate (Kettunen et al., Magn. Reson. Med. 2013; 70: 1200).
A potentially better way to detect response would be to monitor flux through the entire pathway, from glucose to lactate. However, a drawback of the hyperpolarisation technique is that the polarization is relatively short-lived and hitherto we thought that it would not survive passage of the 13C label between glucose and lactate. We have now shown that deuteration of the molecule ([U-2H, U-13C]glucose) extends the lifetime sufficiently that we can detect flux of 13C label through all 10 steps of the glycolytic pathway between glucose and lactate (see Figure 1).
Figure 1. The 1H reference image is a conventional image of tissue water protons and shows the tumour (outlined in white). The 13C chemical shift images were acquired following i.v. injection of hyperpolarised [U-2H, U-13C]glucose and show glucose in the abdomen, however lactate is only present within the tumour, reflecting the high rate of tumour glycolysis and the accumulation of lactate. The images are effectively demonstrate the ‘Warburg effect’. The urea image shows non-polarised urea in an adjacent tube, which was used as a standard. From: Rodrigues et al., Nat Med 2014; Epub 8 Dec 2013
Moreover, this flux is substantially decreased in lymphoma tumours 24 h after chemotherapy (Rodrigues et al., Nat Med. 2014; Epub 8 Dec 2013). We also have some preliminary data showing that we can detect flux of label into the pentose phosphate pathway and therefore the capacity of the tumour cells to resist oxidative stress. The ability of certain cancer cells to resist oxidative stress has been correlated with aggressiveness and drug resistance.
To combat the short lifetime of the polarization we have been examining the possibility of ‘parking’ the polarization in a longer-lived so-called singlet state. Although we have successfully generated this state and observed it in vivo we have yet to demonstrate a significant benefit from doing this (Marco-Rius et al., NMR Biomed 2013; 26: 1696). Polarisation is lost primarily through cross-relaxation between the hyperpolarised 13C spins in the labelled molecule and protons in either the molecule itself or in solvent water. We have demonstrated that cross relaxation with water protons could provide a method for tracking, in a proton image of the body, the passage of a bolus of the labelled material (Marco-Rius et al., Contrast Media Mol. Imag.2013; In press). The method avoids sampling and thus destroying the 13C polarisation and turns the inevitable loss of polarisation due to cross relaxation into a useful signal.
Future directions
Aberrant glycosylation is a hallmark of cancer. We will continue development of a novel method for assessing tumour glycosylation state, in which sugar analogues are incorporated metabolically by tumour cells in vivo and detected subsequently by a highly selective chemical reaction (‘click chemistry’) with an imaging probe (Neves et al., Bioconjug. Chem. 2013; 24: 934 (cover article); Stairs et al., Chembiochem 2013; 14: 1063; Wainman et al., Org. Biomol. Chem. 2013; 11: 7297). We are currently examining whether this method can be used to assess the metastatic potential of tumours.
We will continue development of a targeted radionuclide and fluorophore-labelled imaging agent for detecting cell death, with a view to commercial development in the preclinical arena and also translation to the clinic. We will continue with the development and application of new hyperpolarised 13C labelled metabolic tracers and expect to do the first clinical studies with pyruvate in the Department of Radiology in late 2014.

•    Caldas, Carlos
Breast cancer functional genomics
We have redefined breast cancer as a constellation of 10 genomic driver-based subtypes. This new molecular taxonomy of breast cancer will now be translated into the clinic in stratification, tumour monitoring and therapy studies. In parallel we will continue to develop models to characterize the biology of these subtypes.
Translational breast cancer genomics: applications of molecular profiling in prognosis, prediction and novel therapeutics
The genomic landscapes of breast cancer are dominated by somatic copy number alterations (CNAs), which further supports the significance of the novel molecular taxonomy of breast cancer. We have completed the targeted sequencing at 250-500x depth of 180 genes in all 2,000 cases (and an additional 500) used for the discovery of the 10 integrative clustering subtypes. This reveals distinct patterns of SNVs (single nucleotide variants) across the 10 integrative clusters (Figure 1).
Figure 1. Word clouds illustrating the distributions of mutations in 178 genes in four Integrative Clusters (IntClusts) enriched for ER positive tumours.  In each panel, the size of each word corresponds to the relative frequency of mutations observed in a given gene for that IntClust.
We are now integrating the CNA, SNV and expression data to identify all the drivers across the subtypes. We will also define the patterns of pathway disruption, clonal architecture and clinical correlation. Our aim is to define a minimal set of features that can be used to generate a simple molecular test, ideally performed using routinely collected paraffin-embedded tumour blocks, that can be used prospectively to assign any new tumour to one of the subtypes and identify the driver mutations of the particular tumour. This test can be used for stratification, to design patient-specific tumour monitoring, and ultimately to assign the best therapy to the particular patient.
We have also conducted seminal studies in metastatic breast cancer that have shown that cell-free circulating tumour DNA (ctDNA) in plasma is a better tumour burden biomarker than circulating tumour cells (CTCs). Rises in ctDNA often precede radiological tumour progression by a few months, the dynamic range of ctDNA is several-fold better than CTCs, and the ability to quantify different mutations affords the possibility of non-invasive clonal tracking. In patients with high tumour burden cancer exomes can be directly sequenced in ctDNA in plasma and provide a true liquid biopsy to identify mutations associated with resistance. We are now studying several cases for which we have ctDNA, primary and metastatic tumour biopsies, to characterize the clonal architecture in these unique samples. We have also pilot data that shows promise for ctDNA as an early response biomarker in neoadjuvant therapy and plan to start studies looking at its value as an early relapse biomarker.
We have continued our collaborative efforts to develop image analyses algorithms adapted from astronomy to robustly classify tumour cells, lymphocytes and stromal cells in histological sections of primary human breast cancers. Once this is done individual cells can be treated as objects and both homotypic and heterotypic spatial correlations characterized and integrated with other pathological, biological or clinical features (Figure 2). Our aim is to use these spatial correlations as potential classifiers with prognostic and predictive value. We are also exploring image-processing algorithms to analyse higher-level architectural features as new descriptors of different breast cancer subtypes.
Figure 2. Astronomical image-processing deciphers morphological complexity. Diagram illustrating the process by which spatial correlations are estimated between classified cells. Top panel: A standard histopathological image (left) is subjected to image-processing including detection and classification of cell nuclei utilising a pathologist-trained dataset and k-nearest-neighbour classifier (right). The positions of cells classified as cancer cells (red), stromal cells (beige) and lymphocytes (green) are used to estimate auto-correlation (relationship of each cell type to cells of the same cell type) and cross-correlation (relationship of different cell types to each other). Bottom panel: Line plots depict the pattern of these correlations according to the pixel-distance from each detected cell. Statistics for calculating correlations are borrowed directly from astronomical applications.
We have used our unique tissue microarray (TMA) resource to look at the association between T-cell infiltration and breast cancer survival in 12,439 patients. This has shown that the presence of CD8+ T-cells in breast cancer is associated with a significant reduction in the relative risk of death from the disease in both the ER-negative and the ER-positive HER2-positive subtypes. We have also conducted the largest study of AR as a prognostic marker in breast cancer.
Collaborators: Sam Aparicio (University of British Columbia), Simon Tavaré, Jason Carroll and Florian Markowetz (CRUK CI), Paul Pharoah (Strangeways Research Laboratory), Mike Irwin and Nick Walton (Institute of Astronomy), Richard Baird and Helena Earl (Department of Oncology).
Functional breast cancer genomics: characterising tumour initiating/cancer stem cells in breast cancer subtypes
We have generated a collection of patient-derived tumour xenografts (PDTXs), and have imported others from collaborators, aiming at having a representation of the 10 breast cancer subtypes. All of these PDTXs are being extensively characterized with  whole genome/whole exome sequencing, expression profiling, miRNA profiling and whole genome/reduced-representation bisulfite sequencing. Importantly for all the models generated we have matched normal DNA, and the originating primary tumour or metastasis where a similar molecular profiling is performed. We have now optimised protocols to derive viable single-cell suspensions from each xenograft, which we designate as patient-derived tumour cells (PDTCs). These PDTCs can be used 24 hours after collection for in vitro perturbation (TGFβ exposure, miRNA over-expression, shRNA or RNAi), but crucially also for high-throughput in vitro drug screening. In collaboration with the Sanger Institute we have now conducted pilot experiments screening around 100 compounds in 17 distinct PDTCs, establishing proof of principle that eventually we will be able to completely replace existing cell lines for these experiments. Our aim is to correlate the drug responses/resistance with the molecular profiles of the PDTCs/PDTXs and hence identify novel predictive biomarkers for translational use.
Collaborators: John Stingl (CRUK CI), Sam Aparicio (University of British Columbia), Mathew Garnett and Ultan McDermott (Sanger Institute).

•    Carroll, Jason
Nuclear receptor transcription
We are interested in defining the genomic and molecular features of oestrogen receptor (ER)-mediated transcription in breast cancer cells. We are specifically interested in understanding how these events and the machinery involved cause breast cancer cells to grow.
Oestrogen receptor is the defining feature of luminal breast cancers, where it functions as a transcription factor to induce cell cycle progression. ER is also the target of most endocrine therapies, including tamoxifen and aromatase inhibitors, which are effective treatments. However, some women can develop resistance to these drugs and in many cases, ER simply gets switched back on again, despite the presence of the drug.  ER transcriptional activity requires a number of co-factors and co-operating transcription factors that possess enzymatic activity to alter chromatin structure, the outcome of which determines transcriptional activity. It is currently known that a number of ER co-factors can either assist in transcription (including SRC-1 and AIB-1) or are involved in gene repression by tamoxifen (including N-CoR and SMRT).
In addition, it is now known that ER requires proteins called pioneer factors to be able to maintain its association with DNA. Two of these proteins are FoxA1 and GATA3.  FoxA1 is required for all ER-DNA interactions and in the absence of FoxA1, ER does not associate with DNA, switch genes on or cause cells to grow. Importantly, FoxA1 is also required for growth of cells that have acquired resistance to standard therapies, such as tamoxifen. Therefore, FoxA1 constitutes an attractive drug target for women with drug resistant breast cancer. Recent discoveries show that both GATA3 and FoxA1 are mutated in a significant fraction of women with breast cancer, but we do not currently know what the functional consequences of these changes are.
Characterisation of the role of GATA3 in ER biology
We are interested in identifying and characterising the role of the putative pioneer factor GATA3 in regulating ER activity. Using transcription factor mapping techniques (ChIP-seq), we have identified all the GATA3 binding events in a breast cancer cell line. This reveals high overlap with ER, supporting the hypothesis that GATA3 is intimately involved in ER function. For the first time, we have specifically removed GATA3 and assessed the impact on ER function in breast cancer cells. Unlike FoxA1, which is required for all ER-DNA interactions, we find that loss of GATA3 results in inhibition of some ER binding events, and also results in the reprogramming of novel ER binding events not normally seen in the presence of GATA3. These new ER-DNA interaction regions only observed in the absence of GATA3 correlate with changes in the genes that are regulated by ER, showing that the novel ER binding events are functionally relevant for breast cancer cells. Since GATA3 is mutated in more than 10% of all breast cancers, we believe that alterations in GATA3 sequence fidelity may be impacting ER binding capacity and the target genes that are regulated by ER. We are currently exploring what specific impact GATA3 mutations have on breast cancer cell growth and drug response.
Genomic analysis of ER function in primary breast cancer
All ER genomic studies to date have been limited to breast cancer cell line models, yet they have revealed extraordinary features about ER biology. We have now been able to extend genomic transcription factor mapping experiments into frozen primary breast cancer samples, by performing ER ChIP-sequencing in luminal breast cancer material. The data confirm that ER ChIP-seq can be performed in primary breast cancer samples and that the ER binding events accurately represent the binding sites in the cell lines. However, there are significant numbers of ER binding events that are acquired in tumours with a poor clinical outcome and in metastatic material that originated from an ER positive breast cancer. The novel ER binding events correlate with genes that have predictive value in independent breast cancer cohorts. We can model these events using drug sensitive or resistant cell line models, where ER binding events are dynamic and can be reprogrammed with growth factor stimulation. The reprogrammed ER binding events are mediated by changes in the pioneer factor FoxA1. We are currently exploring what enables changes in FoxA1, since these mediate the changes in ER binding events and subsequently influence the transcriptome. In addition, in close collaboration with the Caldas laboratory, we are embarking on a large scale transcription factor mapping experiment to identify ER and FoxA1 binding events by ChIP-seq, in ~200 primary breast cancers with detailed transcriptomic information and clinical follow up.
Understanding the role of androgen receptor in breast cancer
We recently showed that a subset of breast cancers, called molecular apocrine tumours, are driven by the male hormone receptor androgen receptor (AR) instead of ER. Unexpectedly, AR simply substitutes for ER and gets recruited to the same sites in the genome and subsequently regulates the same genes normally switched on by ER.
Figure 1. A collaboration between the Carroll and Neal laboratories has provided insight into how the male protein androgen receptor (AR) functions in breast cancer. Protein-DNA mapping experiments showed that in breast cancer cells, androgen receptor behaves less like it does in prostate cancer and instead it mimics the oestrogen receptor (ER). The data show a region of a chromosome and the binding sites of AR in the breast (purple) and prostate cancer (blue) contexts and ER in the breast cancer cells (red).
We had also made the observation that FoxA1 was mediating AR-DNA interactions, which paralleled the events normally seen between FoxA1 and ER. More recently we have explored the requirement for FoxA1 in AR-DNA binding capacity in molecular apocrine breast cancer cells. We find that specific loss of FoxA1 results in a reprogramming of AR to new regions in the genome. This is similar to what is observed in prostate cancer models and also what we observed between GATA3 and ER in breast cancer. Therefore, in molecular apocrine breast cancer, AR can mimic ER to regulate the genes normally controlled by ER, but its ability to move around the genome in the absence of FoxA1 is closer to what is observed in the prostate context. We are currently exploring the underlying mechanisms that govern AR reprogramming in the absence of FoxA1 and the potential impact that FoxA1 mutations may have on this process in breast cancer.


URGENT INFORMATION: This is to inform the general public that venue for the 2018 induction ceremony has been changed from the Novella Planet Hotel, Port Novo, Republic of Benin to LTV hall.  The new venue for the induction ceremony of our prestigious and reputable international professional bodies shall be Lagos State Television Combo Hall, Agidingbi, Ikeja, Lagos, Nigeria.  

Time :  12 Noon.     
Date :  May 12th,  2018.  Your presence would be highly appreciated sir/ma.