Publications by authors named "Yaoyong Li"

27 Publications

  • Page 1 of 1

The forkhead transcription factor FOXK2 premarks lineage-specific genes in human embryonic stem cells for activation during differentiation.

Nucleic Acids Res 2021 02;49(3):1345-1363

Faculty of Biology, Medicine and Health, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.

Enhancers play important roles in controlling gene expression in a choreographed spatial and temporal manner during development. However, it is unclear how these regulatory regions are established during differentiation. Here we investigated the genome-wide binding profile of the forkhead transcription factor FOXK2 in human embryonic stem cells (ESCs) and downstream cell types. This transcription factor is bound to thousands of regulatory regions in human ESCs, and binding at many sites is maintained as cells differentiate to mesendodermal and neural precursor cell (NPC) types, alongside the emergence of new binding regions. FOXK2 binding is generally associated with active histone marks in any given cell type. Furthermore newly acquired, or retained FOXK2 binding regions show elevated levels of activating histone marks following differentiation to NPCs. In keeping with this association with activating marks, we demonstrate a role for FOXK transcription factors in gene activation during NPC differentiation. FOXK2 occupancy in ESCs is therefore an early mark for delineating the regulatory regions, which become activated in later lineages.
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http://dx.doi.org/10.1093/nar/gkaa1281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897486PMC
February 2021

Cooperative behaviour and phenotype plasticity evolve during melanoma progression.

Pigment Cell Melanoma Res 2020 09 20;33(5):695-708. Epub 2020 Mar 20.

Manchester Cancer Research Centre, Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

A major challenge for managing melanoma is its tumour heterogeneity based on individual co-existing melanoma cell phenotypes. These phenotypes display variable responses to standard therapies, and they drive individual steps of melanoma progression; hence, understanding their behaviour is imperative. Melanoma phenotypes are defined by distinct transcriptional states, which relate to different melanocyte lineage development phases, ranging from a mesenchymal, neural crest-like to a proliferative, melanocytic phenotype. It is thought that adaptive phenotype plasticity based on transcriptional reprogramming drives melanoma progression, but at which stage individual phenotypes dominate and moreover, how they interact is poorly understood. We monitored melanocytic and mesenchymal phenotypes throughout melanoma progression and detected transcriptional reprogramming at different stages, with a gain in mesenchymal traits in circulating melanoma cells (CTCs) and proliferative features in metastatic tumours. Intriguingly, we found that distinct phenotype populations interact in a cooperative manner, which generates tumours of greater "fitness," supports CTCs and expands organotropic cues in metastases. Fibronectin, expressed in mesenchymal cells, acts as key player in cooperativity and promotes survival of melanocytic cells. Our data reveal an important role for inter-phenotype communications at various stages of disease progression, suggesting these communications could act as therapeutic target.
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http://dx.doi.org/10.1111/pcmr.12873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496243PMC
September 2020

Geno2proteo, a Tool for Batch Retrieval of DNA and Protein Sequences from Any Genomic or Protein Regions.

J Integr Bioinform 2019 Jul 13;16(3). Epub 2019 Jul 13.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.

The interconversion of sequences that constitute the genome and the proteome is becoming increasingly important due to the generation of large amounts of DNA sequence data. Following mapping of DNA segments to the genome, one fundamentally important task is to find the amino acid sequences which are coded within a list of genomic sections. Conversely, given a series of protein segments, an important task is to find the genomic loci which code for a list of protein regions. To perform these tasks on a region by region basis is extremely laborious when a large number of regions are being studied. We have therefore implemented an R package geno2proteo which performs the two mapping tasks and subsequent sequence retrieval in a batch fashion. In order to make the tool more accessible to users, we have created a web interface of the R package which allows the users to perform the mapping tasks by going to the web page http://sharrocksresources.manchester.ac.uk/tofigaps and using the web service.
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http://dx.doi.org/10.1515/jib-2018-0090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798850PMC
July 2019

Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia.

Cell Rep 2018 03;22(13):3641-3659

Leukaemia Biology Laboratory, Cancer Research UK Manchester Institute, The University of Manchester, Manchester Cancer Research Centre Building, 555 Wilmslow Road, Manchester M20 4GJ, UK. Electronic address:

Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.
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http://dx.doi.org/10.1016/j.celrep.2018.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896174PMC
March 2018

Open chromatin profiling identifies AP1 as a transcriptional regulator in oesophageal adenocarcinoma.

PLoS Genet 2017 Aug 31;13(8):e1006879. Epub 2017 Aug 31.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.

Oesophageal adenocarcinoma (OAC) is one of the ten most prevalent forms of cancer and is showing a rapid increase in incidence and yet exhibits poor survival rates. Compared to many other common cancers, the molecular changes that occur in this disease are relatively poorly understood. However, genes encoding chromatin remodeling enzymes are frequently mutated in OAC. This is consistent with the emerging concept that cancer cells exhibit reprogramming of their chromatin environment which leads to subsequent changes in their transcriptional profile. Here, we have used ATAC-seq to interrogate the chromatin changes that occur in OAC using both cell lines and patient-derived material. We demonstrate that there are substantial changes in the regulatory chromatin environment in the cancer cells and using this data we have uncovered an important role for ETS and AP1 transcription factors in driving the changes in gene expression found in OAC cells.
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http://dx.doi.org/10.1371/journal.pgen.1006879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578490PMC
August 2017

EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas.

PLoS One 2017 21;12(7):e0181902. Epub 2017 Jul 21.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.

Long non-coding RNAs are being increasingly recognised as important molecules involved in regulating a diverse array of biological functions. For example, many long non-coding RNAs have been associated with tumourigenesis and in this context their molecular functions often involves impacting on chromatin and transcriptional control processes. One important cellular control system that is often deregulated in cancer cells is the ERK MAP kinase pathway. Here we have investigated whether ERK pathway signaling in response to EGF stimulation, leads to changes in the production of long non-coding RNAs. We identify several different classes of EGF pathway-regulated lncRNAs. We focus on one of the inducible lincRNAs, EGF inducible long intergenic non-coding RNA 1 (EINCR1). EINCR1 is predominantly nuclear and shows delayed activation kinetics compared to other immediate-early EGF-inducible genes. In humans it is expressed in a tissue-specific manner and is mainly confined to the heart but it exhibits little evolutionary conservation. Importantly, in several cancers EINCR1 shows elevated expression levels which correlate with poor survival in lung adenocarcinoma patients. In the context of lung adenocarcinomas, EINCR1 expression is anti-correlated with the expression of several protein coding EGF-regulated genes. A potential functional connection is demonstrated as EINCR1 overexpression is shown to reduce the expression of EGF-regulated protein coding genes including FOS and FOSB.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181902PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521836PMC
September 2017

Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemosensitive and chemorefractory small-cell lung cancer.

Nat Med 2017 01 21;23(1):114-119. Epub 2016 Nov 21.

Clinical and Experimental Pharmacology Group, CRUK Manchester Institute, University of Manchester, Manchester, UK.

In most patients with small-cell lung cancer (SCLC)-a metastatic, aggressive disease-the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan-Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.
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http://dx.doi.org/10.1038/nm.4239DOI Listing
January 2017

Jun-Mediated Changes in Cell Adhesion Contribute to Mouse Embryonic Stem Cell Exit from Ground State Pluripotency.

Stem Cells 2016 05 13;34(5):1213-24. Epub 2016 Feb 13.

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

Embryonic stem cells (ESC) are able to give rise to any somatic cell type. A lot is known about how ESC pluripotency is maintained, but comparatively less is known about how differentiation is promoted. Cell fate decisions are regulated by interactions between signaling and transcriptional networks. Recent studies have shown that the overexpression or downregulation of the transcription factor Jun can affect the ESC fate. Here we have focussed on the role of the Jun in the exit of mouse ESCs from ground state pluripotency and the onset of early differentiation. Transcriptomic analysis of differentiating ESCs reveals that Jun is required to upregulate a programme of genes associated with cell adhesion as ESCs exit the pluripotent ground state. Several of these Jun-regulated genes are shown to be required for efficient adhesion. Importantly this adhesion is required for the timely regulated exit of ESCs from ground state pluripotency and the onset of early differentiation events. Stem Cells 2016;34:1213-1224.
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http://dx.doi.org/10.1002/stem.2294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864893PMC
May 2016

Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Mol Oncol 2016 Apr 19;10(4):566-74. Epub 2015 Nov 19.

Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.

Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours. In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit.
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http://dx.doi.org/10.1016/j.molonc.2015.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834815PMC
April 2016

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

Cancer Res 2016 Feb 4;76(3):724-35. Epub 2015 Dec 4.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, United Kingdom.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0701-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740929PMC
February 2016

GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1.

Nat Cell Biol 2016 Jan 30;18(1):21-32. Epub 2015 Nov 30.

CRUK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.
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http://dx.doi.org/10.1038/ncb3276DOI Listing
January 2016

Stromal cell-mediated mitochondrial redox adaptation regulates drug resistance in childhood acute lymphoblastic leukemia.

Oncotarget 2015 Dec;6(40):43048-64

Children's Cancer Group, Institute of Cancer Science, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom.

Despite the high cure rates in childhood acute lymphoblastic leukemia (ALL), relapsed ALL remains a significant clinical problem. Genetic heterogeneity does not adequately explain variations in response to therapy. The chemoprotective tumor microenvironment may additionally contribute to disease recurrence. This study identifies metabolic reprogramming of leukemic cells by bone marrow stromal cells (BMSC) as a putative mechanism of drug resistance. In a BMSC-extracellular matrix culture model, BMSC produced chemoprotective soluble factors and facilitated the emergence of a reversible multidrug resistant phenotype in ALL cells. BMSC environment induced a mitochondrial calcium influx leading to increased reactive oxygen species (ROS) levels in ALL cells. In response to this oxidative stress, drug resistant cells underwent a redox adaptation process, characterized by a decrease in ROS levels and mitochondrial membrane potential with an upregulation of antioxidant production and MCL-1 expression. Similar expanded subpopulations of low ROS expressing and drug resistant cells were identified in pre-treatment bone marrow samples from ALL patients with slower response to therapy. This suggests that the bone marrow microenvironment induces a redox adaptation in ALL subclones that protects against cytotoxic stress and potentially gives rise to minimal residual disease. Targeting metabolic remodeling by inhibiting antioxidant production and antiapoptosis was able to overcome drug resistance. Thus metabolic plasticity in leukemic cell response to environmental factors contributes to chemoresistance and disease recurrence. Adjunctive strategies targeting such processes have the potential to overcome therapeutic failure in ALL.
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http://dx.doi.org/10.18632/oncotarget.5528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767490PMC
December 2015

Evaluation and validation of a robust single cell RNA-amplification protocol through transcriptional profiling of enriched lung cancer initiating cells.

BMC Genomics 2014 Dec 17;15:1129. Epub 2014 Dec 17.

Nucleic Acid Biomarker Laboratory, Clinical & Experimental Pharmacology, CR-UK Manchester Institute, University of Manchester, Manchester M20 4BX, UK.

Background: Although profiling of RNA in single cells has broadened our understanding of development, cancer biology and mechanisms of disease dissemination, it requires the development of reliable and flexible methods. Here we demonstrate that the EpiStem RNA-Amp™ methodology reproducibly generates microgram amounts of cDNA suitable for RNA-Seq, RT-qPCR arrays and Microarray analysis.

Results: Initial experiments compared amplified cDNA generated by three commercial RNA-Amplification protocols (Miltenyi μMACS™ SuperAmp™, NuGEN Ovation® One-Direct System and EpiStem RNA-Amp™) applied to single cell equivalent levels of RNA (25-50 pg) using Affymetrix arrays. The EpiStem RNA-Amp™ kit exhibited the highest sensitivity and was therefore chosen for further testing. A comparison of Affymetrix array data from RNA-Amp™ cDNA generated from single MCF7 and MCF10A cells to reference controls of unamplified cDNA revealed a high degree of concordance. To assess the flexibility of the amplification system single cell RNA-Amp™ cDNA was also analysed using RNA-Seq and high-density qPCR, and showed strong cross-platform correlations. To exemplify the approach we used the system to analyse RNA profiles of small populations of rare cancer initiating cells (CICs) derived from a NSCLC patient-derived xenograft. RNA-Seq analysis was able to identify transcriptional differences in distinct subsets of CIC, with one group potentially enriched for metastasis formation. Pathway analysis revealed that the distinct transcriptional signatures demonstrated in the CIC subpopulations were significantly correlated with published stem-cell and epithelial-mesenchymal transition signatures.

Conclusions: The combined results confirm the sensitivity and flexibility of the RNA-Amp™ method and demonstrate the suitability of the approach for identifying clinically relevant signatures in rare, biologically important cell populations.
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http://dx.doi.org/10.1186/1471-2164-15-1129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320548PMC
December 2014

JNK suppresses tumor formation via a gene-expression program mediated by ATF2.

Cell Rep 2014 Nov 13;9(4):1361-74. Epub 2014 Nov 13.

Department of Cell Regulation, CRUK Manchester Institute, Paterson Building, University of Manchester, Manchester M20 4BX, UK. Electronic address:

JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development.
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http://dx.doi.org/10.1016/j.celrep.2014.10.043DOI Listing
November 2014

Discrepancies in cancer genomic sequencing highlight opportunities for driver mutation discovery.

Cancer Res 2014 Nov 25;74(22):6390-6396. Epub 2014 Sep 25.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, M20 4BX, UK.

Cancer genome sequencing is being used at an increasing rate to identify actionable driver mutations that can inform therapeutic intervention strategies. A comparison of two of the most prominent cancer genome sequencing databases from different institutes (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer) revealed marked discrepancies in the detection of missense mutations in identical cell lines (57.38% conformity). The main reason for this discrepancy is inadequate sequencing of GC-rich areas of the exome. We have therefore mapped over 400 regions of consistent inadequate sequencing (cold-spots) in known cancer-causing genes and kinases, in 368 of which neither institute finds mutations. We demonstrate, using a newly identified PAK4 mutation as proof of principle, that specific targeting and sequencing of these GC-rich cold-spot regions can lead to the identification of novel driver mutations in known tumor suppressors and oncogenes. We highlight that cross-referencing between genomic databases is required to comprehensively assess genomic alterations in commonly used cell lines and that there are still significant opportunities to identify novel drivers of tumorigenesis in poorly sequenced areas of the exome. Finally, we assess other reasons for the observed discrepancy, such as variations in dbSNP filtering and the acquisition/loss of mutations, to give explanations as to why there is a discrepancy in pharmacogenomic studies, given recent concerns with poor reproducibility of data.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247168PMC
November 2014

Epithelial and stromal microRNA signatures of columnar cell hyperplasia linking Let-7c to precancerous and cancerous breast cancer cell proliferation.

PLoS One 2014 14;9(8):e105099. Epub 2014 Aug 14.

Center for Molecular Pathology, Skåne University Hospital, Department of Laboratory Medicine Malmö, Lund University, Malmö, Sweden; Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, Manchester Academic Health Science Centre, Paterson Institute for Cancer Research, The Christie National Health Service Foundation Trust, Manchester, United Kingdom; Sahlgrenska Cancer Center, Department of Biomedicine, University of Gothenburg, Gothenburg, Sweden.

Columnar cell hyperplasia (CCH) is the earliest histologically identifiable breast lesion linked to cancer progression and is characterized by increased proliferation, decreased apoptosis and elevated oestrogen receptor α (ERα) expression. The mechanisms underlying the initiation of these lesions have not been clarified but might involve early and fundamental changes in cancer progression. MiRNAs are key regulators of several biological processes, acting by influencing the post-transcriptional regulation of numerous targets, thus making miRNAs potential candidates in cancer initiation. Here we have defined novel epithelial as well as stromal miRNA signatures from columnar cell hyperplasia lesions compared to normal terminal duct lobular units by using microdissection and miRNA microarrays. Let-7c were among the identified downregulated epithelial miRNAs and its functions were delineated in unique CCH derived cells and breast cancer cell line MCF-7 suggesting anti-proliferative traits potentially due to effects on Myb and ERα. MiR-132 was upregulated in the stroma surrounding CCH compared to stoma surrounding normal terminal duct lobular units (TDLUs), and overexpression of miR-132 in immortalized fibroblasts and in fibroblasts co-cultured with epithelial CCH cells caused substantial expression changes of genes involved in metabolism, DNA damage and cell motility. The miRNA signatures identified in CCH indicate early changes in the epithelial and stromal compartment of CCH and could represent early key alterations in breast cancer progression that potentially could be targeted in novel prevention or treatment schedules.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133372PMC
March 2016

RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence.

Blood 2014 Sep 31;124(11):e11-20. Epub 2014 Jul 31.

Cancer Research UK Stem Cell Biology Group, and.

During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development.
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http://dx.doi.org/10.1182/blood-2014-04-572958DOI Listing
September 2014

Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer.

Nat Med 2014 Aug 1;20(8):897-903. Epub 2014 Jun 1.

1] Clinical and Experimental Pharmacology Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK. [2].

Small-cell lung cancer (SCLC), an aggressive neuroendocrine tumor with early dissemination and dismal prognosis, accounts for 15-20% of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable, and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice, and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged, and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms.
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http://dx.doi.org/10.1038/nm.3600DOI Listing
August 2014

A global non-coding RNA system modulates fission yeast protein levels in response to stress.

Nat Commun 2014 May 23;5:3947. Epub 2014 May 23.

Applied Computational Biology and Bioinformatics Group, Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

Non-coding RNAs (ncRNAs) are frequent and prevalent across the taxa. Although individual non-coding loci have been assigned a function, most are uncharacterized. Their global biological significance is unproven and remains controversial. Here we investigate the role played by ncRNAs in the stress response of Schizosaccharomyces pombe. We integrate global proteomics and RNA sequencing data to identify a systematic programme in which elevated antisense RNA arising both from ncRNAs and from 3'-overlapping convergent gene pairs is directly associated with substantial reductions in protein levels throughout the genome. We describe an extensive array of ncRNAs with trans associations that have the potential to influence multiple pathways. Deletion of one such locus reduces levels of atf1, a transcription factor downstream of the stress-activated mitogen-activated protein kinase (MAPK) pathway, and alters sensitivity to oxidative stress. These non-coding transcripts therefore regulate specific stress responses, adding unanticipated information-processing capacity to the MAPK signalling system.
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http://dx.doi.org/10.1038/ncomms4947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050258PMC
May 2014

Investigation of radiosensitivity gene signatures in cancer cell lines.

PLoS One 2014 22;9(1):e86329. Epub 2014 Jan 22.

Translational Radiobiology Group, The University of Manchester, Manchester, United Kingdom.

Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086329PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899227PMC
November 2014

CXCL12 induces lung cancer cell migration by polarized mtDNA redistribution.

Hum Cell 2014 Jan;27(1):22-8

Instability of mitochondrial DNA (mtDNA) has been associated with the initiation and development of cancer, but the specific role of mtDNA in the invasiveness and migration of cancer cells remains unclear. In this study, we investigated whether the chemokine CXCL12 causes intact mitochondria to redistribute in cancer cells and, in this way, to increase cell invasiveness and migration. A549 lung cancer cells with intact mtDNA (mtDNA+) and ρ(0)A549 cells depleted of mtDNA (mtDNA-) by long-term ethidium bromide incubation were examined for their responses to CXCL12 in a transwell migration assay and for mitochondrial distribution by fluorescence microscopy. Intact A549 cells showed significantly increased migration and increased polar distribution of mitochondria (asymmetry)in response to CXCL12. However, ρ(0)A549 cells showed no changes in mitochondrial distribution in response to CXCL12, and only a few ρ(0)A549 cells migrated across the transwell membrane after CXCL12 treatment. These results demonstrate that, in A549 lung cancer cells, intact mitochondrial DNA is necessary for mitochondrial redistribution and a chemotactic response to CXCL12.
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http://dx.doi.org/10.1007/s13577-013-0077-4DOI Listing
January 2014

Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer.

Proc Natl Acad Sci U S A 2013 Jul 8;110(30):12426-31. Epub 2013 Jul 8.

Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

Approximately 70% of patients with non-small-cell lung cancer present with late-stage disease and have limited treatment options, so there is a pressing need to develop efficacious targeted therapies for these patients. This remains a major challenge as the underlying genetic causes of ~50% of non-small-cell lung cancers remain unknown. Here we demonstrate that a targeted genetic dependency screen is an efficient approach to identify somatic cancer alterations that are functionally important. By using this approach, we have identified three kinases with gain-of-function mutations in lung cancer, namely FGFR4, MAP3K9, and PAK5. Mutations in these kinases are activating toward the ERK pathway, and targeted depletion of the mutated kinases inhibits proliferation, suppresses constitutive activation of downstream signaling pathways, and results in specific killing of the lung cancer cells. Genomic profiling of patients with lung cancer is ushering in an era of personalized medicine; however, lack of actionable mutations presents a significant hurdle. Our study indicates that targeted genetic dependency screens will be an effective strategy to elucidate somatic variants that are essential for lung cancer cell viability.
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http://dx.doi.org/10.1073/pnas.1305207110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725071PMC
July 2013

Enhanced detection of ubiquitin isopeptides using reductive methylation.

J Am Soc Mass Spectrom 2013 Mar 30;24(3):421-30. Epub 2013 Jan 30.

Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, UK.

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. ᅟ
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http://dx.doi.org/10.1007/s13361-012-0538-0DOI Listing
March 2013

Using prior information from the medical literature in GWAS of oral cancer identifies novel susceptibility variant on chromosome 4--the AdAPT method.

PLoS One 2012 25;7(5):e36888. Epub 2012 May 25.

Section of Genetics, International Agency for Research on Cancer (IARC), Lyon, France.

Background: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS.

Methods: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest--the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer.

Results: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5×10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found.

Conclusion: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036888PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360735PMC
December 2012

The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells.

Cancer Cell 2012 Apr 29;21(4):473-87. Epub 2012 Mar 29.

Cancer Research UK Leukaemia Biology Laboratory, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

Using a mouse model of human MLL-AF9 leukemia, we identified the lysine-specific demethylase KDM1A (LSD1 or AOF2) as an essential regulator of leukemia stem cell (LSC) potential. KDM1A acts at genomic loci bound by MLL-AF9 to sustain expression of the associated oncogenic program, thus preventing differentiation and apoptosis. In vitro and in vivo pharmacologic targeting of KDM1A using tranylcypromine analogs active in the nanomolar range phenocopied Kdm1a knockdown in both murine and primary human AML cells exhibiting MLL translocations. By contrast, the clonogenic and repopulating potential of normal hematopoietic stem and progenitor cells was spared. Our data establish KDM1A as a key effector of the differentiation block in MLL leukemia, which may be selectively targeted to therapeutic effect.
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http://dx.doi.org/10.1016/j.ccr.2012.03.014DOI Listing
April 2012

Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe.

Mol Syst Biol 2011 Dec 20;7:559. Epub 2011 Dec 20.

CRUK Applied Computational Biology and Bioinformatics Group, Cancer Research UK, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK.

Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3' termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent 'horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply 'genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe.
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http://dx.doi.org/10.1038/msb.2011.90DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738847PMC
December 2011

A comparison of massively parallel nucleotide sequencing with oligonucleotide microarrays for global transcription profiling.

BMC Genomics 2010 May 5;11:282. Epub 2010 May 5.

Applied Computational Biology and Bioinformatics, Cancer Research UK, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester M20 4BX UK.

Background: RNA-Seq exploits the rapid generation of gigabases of sequence data by Massively Parallel Nucleotide Sequencing, allowing for the mapping and digital quantification of whole transcriptomes. Whilst previous comparisons between RNA-Seq and microarrays have been performed at the level of gene expression, in this study we adopt a more fine-grained approach. Using RNA samples from a normal human breast epithelial cell line (MCF-10a) and a breast cancer cell line (MCF-7), we present a comprehensive comparison between RNA-Seq data generated on the Applied Biosystems SOLiD platform and data from Affymetrix Exon 1.0ST arrays. The use of Exon arrays makes it possible to assess the performance of RNA-Seq in two key areas: detection of expression at the granularity of individual exons, and discovery of transcription outside annotated loci.

Results: We found a high degree of correspondence between the two platforms in terms of exon-level fold changes and detection. For example, over 80% of exons detected as expressed in RNA-Seq were also detected on the Exon array, and 91% of exons flagged as changing from Absent to Present on at least one platform had fold-changes in the same direction. The greatest detection correspondence was seen when the read count threshold at which to flag exons Absent in the SOLiD data was set to t<1 suggesting that the background error rate is extremely low in RNA-Seq. We also found RNA-Seq more sensitive to detecting differentially expressed exons than the Exon array, reflecting the wider dynamic range achievable on the SOLiD platform. In addition, we find significant evidence of novel protein coding regions outside known exons, 93% of which map to Exon array probesets, and are able to infer the presence of thousands of novel transcripts through the detection of previously unreported exon-exon junctions.

Conclusions: By focusing on exon-level expression, we present the most fine-grained comparison between RNA-Seq and microarrays to date. Overall, our study demonstrates that data from a SOLiD RNA-Seq experiment are sufficient to generate results comparable to those produced from Affymetrix Exon arrays, even using only a single replicate from each platform, and when presented with a large genome.
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http://dx.doi.org/10.1186/1471-2164-11-282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877694PMC
May 2010