• Curvature-sensitive kinesin binding can explain microtubule ring formation and reveals chaotic dynamics in a mathematical model.

      Pearce, Simon P; Heil, M; Jensen, O; Jones, G; Prokop, A; School of Mathematics, University of Manchester, Manchester, UK (2018-11)
      Microtubules are filamentous tubular protein polymers which are essential for a range of cellular behaviour, and are generally straight over micron length scales. However, in some gliding assays, where microtubules move over a carpet of molecular motors, individual microtubules can also form tight arcs or rings, even in the absence of crosslinking proteins. Understanding this phenomenon may provide important explanations for similar highly curved microtubules which can be found in nerve cells undergoing neurodegeneration. We propose a model for gliding assays where the kinesins moving the microtubules over the surface induce ring formation through differential binding, substantiated by recent findings that a mutant version of the motor protein kinesin applied in solution is able to lock-in microtubule curvature. For certain parameter regimes, our model predicts that both straight and curved microtubules can exist simultaneously as stable steady states, as has been seen experimentally. Additionally, unsteady solutions are found, where a wave of differential binding propagates down the microtubule as it glides across the surface, which can lead to chaotic motion. Whilst this model explains two-dimensional microtubule behaviour in an experimental gliding assay, it has the potential to be adapted to explain pathological curling in nerve cells.
    • A PAX3/BRN2 rheostat controls the dynamics of BRAF mediated MITF regulation in MITFhigh /AXLlow melanoma.

      Smith, M; Rana, S; Ferguson, J; Rowling, E; Flaherty, K; Wargo, J; Marais, Richard; Wellbrock, C; Manchester Cancer Research Centre, Faculty of Biology, Medicine& Health, Division of Cancer Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT (2018-10-01)
      The BRAF-kinase and the MAPK-pathway are targets of current melanoma therapies. However, MAPK-pathway inhibition results in dynamic changes of down-stream targets that can counteract inhibitor-action not only during treatment, but also in acquired resistant tumours. One such dynamic change involves the expression of the transcription factor MITF, a crucial regulator of cell survival and proliferation in untreated as well as drug-addicted acquired resistant melanoma. Tight control over MITF expression levels is required for optimal melanoma growth, and while it is well established that the MAPK-pathway regulates MITF expression, the actual mechanism is insufficiently understood. We reveal here, how BRAF through action on the transcription factors BRN2 and PAX3 executes control over the regulation of MITF expression in a manner that allows for considerable plasticity. This plasticity provides robustness to the BRAF mediated MITF regulation and explains the dynamics in MITF expression that are observed in patients in response to MAPK-inhibitor therapy. This article is protected by copyright. All rights reserved.
    • Lung colonization by Aspergillus fumigatus is controlled by ZNF77.

      Gago, S; Overton, Nicola L D; Ben-Ghazzi, N; Novak-Frazer, L; Read, N; Denning, D; Bowyer, P; Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, University of Manchester, CTF Building, 46 Grafton Street, Manchester, M13 9NT (2018-09-20)
      Aspergillus fumigatus is a critical pathogen of humans. Exposure to A. fumigatus conidia occurs frequently but is normally cleared from the respiratory airways. In contrast, individuals with respiratory diseases are often highly colonized by fungi. Here, we use genome-edited epithelial cells to show that the genetic variant rs35699176 in ZNF77 causes loss of integrity of the bronchial epithelium and increases levels of extracellular matrix proteins. These changes promote A. fumigatus conidial adhesion, germination and growth. RNA-seq and LC/MS-MS analysis reveal rs35699176 upregulates vesicle trafficking leading to an increment of adhesion proteins. These changes make cells carrying rs35699176 more receptive to A. fumigatus in the early stages of infection. Moreover, patients with fungal asthma carrying rs35699176+/- have higher A. fumigatus loads in their respiratory airway. Our results indicate ZNF77 as a key controller of Aspergillus colonization and suggest its utility as a risk-marker for patient stratification.
    • 'Mind your Moles' study: protocol of a prospective cohort study of melanocytic naevi.

      Koh, U; Janda, M; Aitken, J; Duffy, D; Menzies, S; Sturm, R; Schaider, H; Betz-Stablein, B; Prow, T; Soyer, H; Green, Adèle C; Centre of Health Services Research, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia (2018-09-19)
      Having many melanocytic naevi or 'moles' on the skin is the strongest predictor of melanoma; thus, much can be learnt from investigating naevi in the general population. We aim to improve the understanding of the epidemiology and biology of naevi by conducting a 3-year prospective study of melanocytic naevi in adults.
    • Intratumoral heterogeneity of glioblastoma infiltration revealed by joint histogram analysis of diffusion tensor imaging.

      Li, C; Wang, S; Yan, J; Piper, R; Liu, H; Torheim, Turid; Kim, H; Zou, J; Boonzaier, N; Sinha, R; Matys, T; Markowetz, Florian; Price, S; Cambridge Brain Tumor Imaging Laboratory, Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom (2018-09-17)
      Glioblastoma is a heterogeneous disease characterized by its infiltrative growth, rendering complete resection impossible. Diffusion tensor imaging (DTI) shows potential in detecting tumor infiltration by reflecting microstructure disruption.
    • Revert the SIRT: normalizing SIRT1 activity in myelodysplastic stem cells.

      Simeoni, Fabrizio; Somervaille, Tim C P; Leukaemia Biology Laboratory, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, M20 4JG, UK (2018-09-06)
      Myelodysplastic syndromes are hematologic malignancies with few treatment options and a propensity to transform to acute myeloid leukemia. In this issue of Cell Stem Cell, Sun et al. (2018) report that low SIRT1 levels in myelodysplastic stem cells contribute to aberrant self-renewal through enabling hyperacetylation and reduced activity of TET2.
    • Genomic classifier for guiding treatment of intermediate-risk prostate cancers to dose-escalated image-guided radiotherapy without hormone therapy.

      Berlin, A; Murgic, J; Hosni, A; Pintilie, M; Salcedo, A; Fraser, M; Kamel-Reid, S; Zhang, J; Wang, Q; Ch'ng, C; Deheshi, S; Davicioni, E; van der Kwast, T; Boutros, P; Bristow, Robert G; Chua, M; Princess Margaret Cancer Centre, University Health Network, Ontario, Canada (2018-08-28)
      The NCCN has recently endorsed the stratification of intermediate-risk prostate cancer (IR-PCa) into favorable and unfavourable subgourps, and recommend the addition of androgen deprivation therapy (ADT) to radiotherapy (RT) for unfavorable IR-PCa. Recently, more accurate prognostication was demonstrated by integrating a 22-feature genomic classifier (GC) to the NCCN stratification system. Here, we test the utility of the GC to better identify IR-PCa patients who are sufficiently treated by RT alone.
    • Publisher correction: genomic instability in mutant p53 cancer cells upon entotic engulfment.

      Mackay, H; Moore, D; Hall, Callum; Birkbak, N; Jamal-Hanjani, M; Karim, S; Phatak, V; Piñon, L; Morton, J; Swanton, C; Le Quesne, J; Muller, Patricia; MRC Toxicology Unit, Lancaster Road, LE1 9HN, Leicester, UK (2018-08-28)
      The original version of this article incorrectly omitted an affiliation of Patricia A. J. Muller: 'Cancer Research UK Manchester Institute, The University of Manchester | Alderley Park, Manchester, SK10 4TG, UK'. This has been corrected in both the PDF and HTML versions of the Article.
    • Combined inhibition of PI3Kβ and mTOR inhibits growth of PTEN null tumours.

      Lynch, J; Polanska, U; Hancox, U; Delpuech, O; Maynard, J; Trigwell, Catherine B; Eberlein, Catherine; Lenaghan, C; Polanski, R; Avivar-Valderas, A; Cumberbatch, M; Klinowska, T; Critchlow, S; Cruzalegui, F; Barry, S; IMED Oncology, Bioscience, AstraZeneca (United Kingdom) (2018-08-10)
      Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signalling, however efficacy is often limited by sub-optimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signalling in PTEN null tumors, the PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ and mTOR gave the most effective anti-proliferative effects across a panel of PTEN null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN null tumor models of TNBC, prostate and renal cancers. In vitro the combination resulted in increased suppression of pNDRG1, p4EBP1 as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kβ inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN null tumors.
    • DNA methylation and repressive histones in the promoters of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 genes in human colorectal cancer.

      Sasidharan Nair, V; Toor, S; Taha, R; Shaath, H; Elkord, Eyad; Cancer Research Center, Qatar Biomedical Research Institute, College of Science and Engineering, Hamad Bin Khalifa University, Qatar Foundation, P.O. Box 5825, Doha, Qatar (2018-08-06)
      Colorectal cancer (CRC) is the third most commonly diagnosed human malignancy worldwide. Upregulation of inhibitory immune checkpoints by tumor-infiltrating immune cells (TIICs) or their ligands by tumor cells leads to tumor evasion from host immunosurveillance. Changes in DNA methylation pattern and enrichment of methylated histone marks in the promoter regions could be major contributors to the upregulation of immune checkpoints (ICs) in the tumor microenvironment (TME).
    • Genomic instability in mutant p53 cancer cells upon entotic engulfment.

      Mackay, H; Moore, D; Hall, Callum; Birkbak, N; Jamal-Hanjani, M; Karim, S; Phatak, V; Piñon, L; Morton, J; Swanton, C; Le Quesne, J; Muller, P; MRC Toxicology Unit, Lancaster Road, Leicester, LE1 9HN, UK (2018-08-03)
      Cell-in-cell (CIC) structures are commonly seen in tumours. Their biological significance remains unclear, although they have been associated with more aggressive tumours. Here we report that mutant p53 promotes CIC via live cell engulfment. Engulfed cells physically interfere in cell divisions of host cells and for cells without p53 this leads to host cell death. In contrast, mutant p53 host cells survive, display aberrant divisions, multinucleation and tripolar mitoses. In xenograft studies, CIC-rich p53 mutant/null co-cultures show enhanced tumour growth. Furthermore, our results show that CIC is common within lung adenocarcinomas, is an independent predictor of poor outcome and disease recurrence, is associated with mutant p53 expression and correlated to measures of heterogeneity and genomic instability. These findings suggest that pro-tumorigenic entotic engulfment activity is associated with mutant p53 expression, and the two combined are a key factor in genomic instability.
    • MST2 kinase suppresses rDNA transcription in response to DNA damage by phosphorylating nucleolar histone H2B.

      Pefani, D; Tognoli, M; Pirincci Ercan, D; Gorgoulis, Vassilis G; O'Neill, E; CRUK/MRC Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford (2018-08-01)
      The heavily transcribed rDNA repeats that give rise to the ribosomal RNA are clustered in a unique chromatin structure, the nucleolus. Due to its highly repetitive nature and transcriptional activity, the nucleolus is considered a hotspot of genomic instability. Breaks in rDNA induce a transient transcriptional shut down to conserve energy and promote rDNA repair; however, how nucleolar chromatin is modified and impacts on rDNA repair is unknown. Here, we uncover that phosphorylation of serine 14 on histone H2B marks transcriptionally inactive nucleolar chromatin in response to DNA damage. We identified that the MST2 kinase localises at the nucleoli and targets phosphorylation of H2BS14p in an ATM-dependent manner. We show that establishment of H2BS14p is necessary for damage-induced rDNA transcriptional shut down and maintenance of genomic integrity. Ablation of MST2 kinase, or upstream activators, results in defective establishment of nucleolar H2BS14p, perturbed DNA damage repair, sensitisation to rDNA damage and increased cell lethality. We highlight the impact of chromatin regulation in the rDNA damage response and targeting of the nucleolus as an emerging cancer therapeutic approach.
    • Smoking and cutaneous melanoma: findings from the QSkin sun and health cohort study.

      Dusingize, J; Olsen, C; Pandeya, N; Thompson, B; Webb, P; Green, Adèle C; Neale, R; Whiteman, D; Department of Population Health, QIMR Berghofer Medical Research Institute, Queensland, Australia (2018-08)
      Background: Previous studies suggest that smokers have lower risks of cutaneous melanoma than nonsmokers, but data from population-based prospective studies are scarce. We investigated associations between smoking and melanoma in a cohort study purpose-designed to investigate skin cancer outcomes.Methods: Participants with no prior history of melanoma (n = 38,697) completed a risk factor survey at baseline (2011). Patients were followed through linkage to the cancer registry. We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for associations between smoking (including intensity, duration, time since quitting) and melanoma using multivariate Cox proportional hazards regression, accounting for death as a competing event.Results: During a mean follow-up of 3.5 years, invasive melanomas developed in 247 participants. Compared with never smokers, former smokers (but not current smokers) had lower risks of invasive melanoma (HR 0.76; 95% CI, 0.57-1.01). Among former smokers, risks were lower with greater quantity of cigarettes smoked (HR 0.75; 95% CI, 0.56-0.98 per 10 cigarettes/day). No association was observed with duration of smoking while longer time since quitting was associated with a relative risk of melanoma that was not significantly different from the null (HR 1.18; 95% CI, 0.91-1.51, for every 10 years since quitting).Conclusions: We observed complex associations between smoking and melanoma, with some suggestion that former smokers had lower risks than never or current smokers. The apparent inverse association among former smokers may be due to residual confounding, although surveillance bias or biological effects cannot be excluded entirely.Impact: Smoking does not increase the risk of cutaneous melanoma. Cancer Epidemiol Biomarkers Prev; 27(8); 874-81. ©2018 AACR.
    • Ex vivo expanded tumour-infiltrating lymphocytes from ovarian cancer patients release anti-tumour cytokines in response to autologous primary ovarian cancer cells.

      Owens, Gemma L; Price, M; Cheadle, Eleanor J; Hawkins, Rob; Gilham, David E; Edmondson, R; Gynaecological Oncology, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK (2018-07-23)
      Epithelial ovarian cancer (EOC) is the leading cause of gynaecological cancer-related death in Europe. Although most patients achieve an initial complete response with first-line treatment, recurrence occurs in more than 80% of cases. Thus, there is a clear unmet need for novel second-line treatments. EOC is frequently infiltrated with T lymphocytes, the presence of which has been shown to be associated with improved clinical outcomes. Adoptive T-cell therapy (ACT) using ex vivo-expanded tumour-infiltrating lymphocytes (TILs) has shown remarkable efficacy in other immunogenic tumours, and may represent a promising therapeutic strategy for EOC. In this preclinical study, we investigated the efficacy of using anti-CD3/anti-CD28 magnetic beads and IL-2 to expand TILs from freshly resected ovarian tumours. TILs were expanded for up to 3 weeks, and then subjected to a rapid-expansion protocol (REP) using irradiated feeder cells. Tumours were collected from 45 patients with EOC and TILs were successfully expanded from 89.7% of biopsies. Expanded CD4+ and CD8+ subsets demonstrated features associated with memory phenotypes, and had significantly higher expression of key activation and functional markers than unexpanded TILs. Expanded TILs produced anti-tumour cytokines when co-cultured with autologous tumour cells, inferring tumour cytotoxicity. Our findings demonstrate that it is possible to re-activate and expand tumour-reactive T cells from ovarian tumours. This presents a promising immunotherapy that could be used sequentially or in combination with current therapeutic strategies.
    • Management of organ transplant recipients attending a high-throughput skin cancer surgery and surveillance clinic in Queensland.

      Papier, K; Gordon, L G; Khosrotehrani, K; Isbel, N; Campbell, S; Griffin, A; Green, Adèle C; QIMR Berghofer Medical Research Institute, Population Health Department, Brisbane, Queensland, Australia (2018-07-13)
      The incidence of skin cancer in organ transplant recipients (OTRs) is very high due mainly to long-term immunosuppressive therapy. The problem is particularly severe for organ transplant recipients living in Queensland, Australia, resulting in significant mortality.
    • Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease.

      Tumbale, P; Schellenberg, M; Mueller, G; Fairweather, Emma E; Watson, Mandy; Little, J; Krahn, J; Waddell, Ian D; London, R; Williams, R; Genome Integrity and Structural Biology Laboratory, Department of Health and Human Services, National Institute of Environmental Health Sciences, US National Institutes of Health, Research Triangle Park, NC, USA (2018-07-13)
      The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X-ray structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes POLβ and FEN1. Our results uncover a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X-ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.
    • Crosstalk between Notch, HIF-1α and GPER in breast cancer EMT.

      De Francesco, Ernestina M; Maggiolini, M; Musti, A; Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy. (2018-07-10)
      The Notch signaling pathway acts in both physiological and pathological conditions, including embryonic development and tumorigenesis. In cancer progression, diverse mechanisms are involved in Notch-mediated biological responses, including angiogenesis and epithelial-mesenchymal-transition (EMT). During EMT, the activation of cellular programs facilitated by transcriptional repressors results in epithelial cells losing their differentiated features, like cell⁻cell adhesion and apical⁻basal polarity, whereas they gain motility. As it concerns cancer epithelial cells, EMT may be consequent to the evolution of genetic/epigenetic instability, or triggered by factors that can act within the tumor microenvironment. Following a description of the Notch signaling pathway and its major regulatory nodes, we focus on studies that have given insights into the functional interaction between Notch signaling and either hypoxia or estrogen in breast cancer cells, with a particular focus on EMT. Furthermore, we describe the role of hypoxia signaling in breast cancer cells and discuss recent evidence regarding a functional interaction between HIF-1α and GPER in both breast cancer cells and cancer-associated fibroblasts (CAFs). On the basis of these studies, we propose that a functional network between HIF-1α, GPER and Notch may integrate tumor microenvironmental cues to induce robust EMT in cancer cells. Further investigations are required in order to better understand how hypoxia and estrogen signaling may converge on Notch-mediated EMT within the context of the stroma and tumor cells interaction. However, the data discussed here may anticipate the potential benefits of further pharmacological strategies targeting breast cancer progression.
    • WDR5 modulates cell motility and morphology and controls nuclear changes induced by a 3D environment.

      Wang, Pengbo; Dreger, M; Madrazo, E; Williams, C; Samaniego, R; Hodson, N; Monroy, F; Baena, Esther; Sánchez-Mateos, P; Hurlstone, A; Redondo-Muñoz, J; Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, School of Medical Sciences, Division of Cell Matrix Biology and Regenerative Medicine, The University of Manchester, M13 9PT Manchester, (2018-07-09)
      Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.
    • Early stage NSCLC - challenges to implementing ctDNA-based screening and MRD detection.

      Abbosh, Christopher; Birkbak, Nicolai J; Swanton, Charles; Cancer Research UK Lung Cancer Centre of Excellence London and Manchester, University College London Cancer Institute, London, UK (2018-07-03)
      Circulating tumour DNA (ctDNA) refers to the fraction of cell-free DNA in a patient's blood that originates from a tumour. Advances in DNA sequencing technologies and our understanding of the molecular biology of tumours have resulted in increased interest in exploiting ctDNA as a tool to facilitate earlier detection of cancer and thereby improve therapeutic outcomes by enabling early intervention. ctDNA analysis might also have utility in the adjuvant therapeutic setting by enabling the identification of patients at a high risk of disease recurrence on the basis of the detection of post-surgical minimal (or molecular) residual disease (MRD). This approach could provide the capability to adapt clinical trials in the adjuvant setting in order to optimize risk stratification, and we argue that this objective is achievable with current technologies. Herein, we evaluate contemporary next-generation sequencing (NGS) approaches to ctDNA detection with a focus on non-small-cell lung cancer. We explain the technical and analytical challenges to low-frequency mutation detection using NGS-based ctDNA profiling and evaluate the feasibility of ctDNA profiling in both screening and MRD assessment contexts.
    • Differential prognostic impact of MYC and PCAT-1 copy number alterations and mRNA expression in intermediate risk prostate cancer: Experience from the Canadian Prostate Cancer Gene Consortium cohort

      Bellur, S; Weiss, J; Pintilie, M; Frazer, M; Vincent, H; Boutros, P; Bristow, Robert G; Van der Kwast, T; Toronto Gen Hosp, Toronto, ON, Canada (2018-06-30)