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 (cH2AX 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 Abbreviations 53BP1, p53-binding protein 1; ATCC, American Type Culture Collection; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutated and Rad3 related; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2; COX-2, cyclooxygenase-2; DDR, DNA damage response; FBS, fetal bovine serum; FISH, fluorescence in situ hybridization; GFAP, glial fibrillary acidic protein; HCMV, human cytomegalovirus; IDH, isocitrate dehydrogenase; IE72, immediate early 72; Ini-1, integrase interactor 1; MAP-2, microtubule-associated protein 2; MDC1, mediator of DNA damage checkpoint protein 1; MOI, multiplicity of infection; NGS, next-generation sequencing; Olig2, oligodendrocyte transcription factor 2; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SHH, sonic hedgehog; WNT, wingless; γH2AX, phosphorylated histone H2AX. 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 ...

Around and beyond 53BP1 Nuclear Bodies

“…This led the authors to suggest that the formation of 53BP1 NBs could be a strategy for the viruses to subvert the cell mechanism in order to expand the host cells lifespan and sustain the viral infection. Finally, 53BP1 NBs are also observed in medulloblastoma cells and denote the chronic endogenous replication stress existing in these cells, often infected with human cytomegalovirus [ 76 ]. Remarkably, 53BP1 NBs are mainly observed in a non cell-autonomous manner, that is to say in tumor cells that are not infected with the virus but are close to infected cells, within the tumor.…”

“…Resolving RS is crucial to maintain genome stability and DDR constitutes a major anti-tumor barrier [ 74 , 75 , 76 ]. Interestingly, unperturbed growing medulloblastoma cell lines or sample from patients prior to therapy display elevated 53BP1 NBs, scars of RS in the previous cell cycle [ 76 ]. ATR, the master mediator in response to RS, controls the replication fork progression, prevents its collapse, inhibits new origins firing and assists the restart of stalled forks.…”

Increased cytomegalovirus replication by 5-Azacytidine and viral-induced cytoplasmic expression of DNMT‑1 in medulloblastoma and endothelial cells

“…During recent years HCMV protein and nucleic acid have been detected in several types of tumor tissues, including glioblastoma, neuroblastoma and medulloblastoma (19)(20)(21)(22). HCMV is a widespread β-herpesvirus carried by 60-95% of adults worldwide in both developing and developed countries and establishes a lifelong latency and persistence in myeloid lineage cells with periodic reactivation (23).…”

Protective Mechanisms Against DNA Replication Stress in the Nervous System

“…Glioblastoma, one of the most lethal human cancers, medulloblastoma, the most common pediatric brain tumor, and the pediatric cancer retinoblastoma are among the most studied CNS tumors [108][109][110][111]. The progressions of these CNS tumors depends on unrestricted cell proliferation, and replication stress response pathways are often deregulated in these diseases [112][113][114]. Absent or defective checkpoints contribute to enhanced cell proliferation, the prevention of apoptosis, and other relevant transformation processes.…”