title: Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas.
abstract: Medulloblastomas are the most common, and often fatal, paediatric brain tumours that feature high genomic instability, frequent infection by human cytomegalovirus (HCMV) and resistance to radiation and chemotherapy. The causes of the pronounced chromosomal instability and its potential links with HCMV infection and/or resistance to genotoxic therapies remain largely unknown. To address these issues, here we have combined immunohistochemical analysis of a series of 25 paediatric medulloblastomas, complemented by medulloblastoma cell culture models including experimental HCMV infection. Using eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (γH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour suppressor activation, across our medulloblastoma cohort. Most tumours showed high proliferation (Ki67 marker), variable oxidative DNA damage (8-oxoguanine lesions) and formation of 53BP1 nuclear 'bodies', the latter indicating (along with ATR-Chk1 signalling) endogenous replication stress. The bulk of the clinical specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing responses of normal cells and medulloblastoma cells to HCMV infection, including differential subcellular mislocalization and enhancement of replication stress-related 53BP1 body formation, the latter in cell-non-autonomous manner. Overall, our results strongly indicate that in human medulloblastomas, the DDR checkpoint barrier is widely activated, at least in part due to replication stress. Furthermore, we propose that unorthodox p53 defects other than mutations may allow high proliferation despite the ongoing checkpoint signalling and that the highly prevalent HCMV may impact the medulloblastoma host cell replication stress and DNA repair. Collectively, the scenario we report here likely fuels genomic instability and evolution of medulloblastoma resistance to standard-of-care genotoxic treatments.
authors.affiliation: Department of Medicine, Unit of Microbial Pathogenesis, Karolinska Institutet, Stockholm, Sweden.|Danish Cancer Society Research Center, Copenhagen, Denmark.|Department of Neurosurgery, Karolinska University Hospital, Stockholm, Sweden.|Department of Neurosurgery, Copenhagen University Hospital Rigshospitalet, Denmark.
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.affiliation: Department of Medicine, Unit of Microbial Pathogenesis, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Olesja
authors.initials: O
authors.lastname: Fornara
authors.affiliation: Danish Cancer Society Research Center, Copenhagen, Denmark.
authors.forename: Joanna Maria
authors.initials: JM
authors.lastname: Merchut-Maya
authors.affiliation: Danish Cancer Society Research Center, Copenhagen, Denmark.
authors.forename: Apolinar
authors.initials: A
authors.lastname: Maya-Mendoza
authors.affiliation: Department of Medicine, Unit of Microbial Pathogenesis, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Afshar
authors.initials: A
authors.lastname: Rahbar
authors.affiliation: Department of Medicine, Unit of Microbial Pathogenesis, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Stragliotto
authors.affiliation: Department of Pathology, Copenhagen University Hospital Rigshospitalet, Denmark.
authors.forename: Helle
authors.initials: H
authors.lastname: Broholm
authors.affiliation: Department of Neurosurgery, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Svensson
authors.affiliation: Department of Paediatrics and Adolescent Medicine, Copenhagen University Hospital Rigshospitalet, Denmark.
authors.forename: Astrid
authors.initials: A
authors.lastname: Sehested
authors.affiliation: Department of Medicine, Unit of Microbial Pathogenesis, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Söderberg Naucler
authors.affiliation: Danish Cancer Society Research Center, Copenhagen, Denmark.|Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Karolinska Institute, Stockholm, Sweden.
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.affiliation: Danish Cancer Society Research Center, Copenhagen, Denmark.|Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Karolinska Institute, Stockholm, Sweden.
authors.forename: Jirina
authors.initials: J
authors.lastname: Bartkova
mesh_terms: D000293:Adolescent; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002528:Cerebellar Neoplasms / Q000378:metabolism* / Q000473:pathology; D002648:Child; D002675:Child, Preschool; D003587:Cytomegalovirus / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000378:metabolism* / Q000473:pathology; D004249:DNA Damage; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008297:Male; D008527:Medulloblastoma / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins
keywords: 53BP1, ATM, ATR and p53 tumour suppressors; brain tumours; cytomegalovirus proteins; endogenous DNA damage signalling; oxidative stress; replication stress