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DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1+/− mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs-/-/Ptch1+/− mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.

Background Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. Methods RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. Results RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. Conclusions We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults. Electronic supplementary material The online version of this article (10.1186/s13046-019-1293-x) contains supplementary material, which is available to authorized users.

// Mirella Tanori 1 , Arianna Casciati 1 , Francesco Berardinelli 2 , Simona Leonardi 1 , Emanuela Pasquali 1 , Francesca Antonelli 1 , Barbara Tanno 1 , Paola Giardullo 2,3 , Alessandro Pannicelli 4 , Gabriele Babini 5 , Ilaria De Stefano 3 , Antonella Sgura 2 , Mariateresa Mancuso 1 , Anna Saran 1 and Simonetta Pazzaglia 1 1 Laboratory of Biomedical Technologies, Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico Sostenibile (ENEA), CR-Casaccia, Rome, Italy 2 Department of Science, University Roma Tre, Rome, Italy 3 Department of Radiation Physics, Universita degli Studi Guglielmo Marconi, Rome, Italy 4 Technical Unit of Energetic Efficiency, ENEA, Rome, Italy 5 Department of Physics, University of Pavia, Pavia, Italy Correspondence to: Simonetta Pazzaglia, email: // Keywords : cerebellum, expression profiles, medulloblastoma, apoptosis, senescence Received : June 14, 2016 Accepted : June 26, 2016 Published : July 07, 2016 Abstract Mutations in DNA repair pathways are frequent in human cancers. Hence, gaining insights into the interaction of DNA repair genes is key to development of novel tumor-specific treatment strategies. In this study, we tested the functional relationship in development and oncogenesis between the homologous recombination (HR) factor Rad54 and Parp-1 , a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. We introduced single or combined Rad54 and Parp-1 inactivating germline mutations in Ptc1 heterozygous mice, a well-characterized model of medulloblastoma, the most common malignant pediatric brain tumor. Our study reveals that combined inactivation of Rad54 and Parp-1 causes a marked growth delay culminating in perinatallethality, providing for the first time evidence of synthetic lethal interactions between Rad54 and Parp-1 in vivo . Although the double mutation hampered investigation of Rad54 and Parp-1 interactions in cerebellum tumorigenesis, insights were gained by showing accumulation of endogenous DNA damage and increased apoptotic rate in granule cell precursors (GCPs). A network-based approach to detect differential expression of DNA repair genes in the cerebellum revealed perturbation of p53 signaling in Rad54 -/- / Parp-1 -/- / Ptc1 +/- , and MEFs from combined Rad54/Parp-1 mutants showed p53/p21-dependent typical senescent features. These findings help elucidate the genetic interplay between Rad54 and Parp-1 by suggesting that p53/p21-mediated apoptosis and/or senescence may be involved in synthetic lethal interactions occurring during development and inhibition of tumor growth.

Neural stem cells are highly susceptible to radiogenic DNA damage, however, little is known about their mechanisms of DNA damage response (DDR) and the long-term consequences of genotoxic exposure. Patched1 heterozygous mice (Ptc1+/−) provide a powerful model of medulloblastoma (MB), a frequent pediatric tumor of the cerebellum. Irradiation of newborn Ptc1+/− mice dramatically increases the frequency and shortens the latency of MB. In this model, we investigated the mechanisms through which multipotent neural progenitors (NSCs) and fate-restricted progenitor cells (PCs) of the cerebellum respond to DNA damage induced by radiation, and the long-term developmental and oncogenic consequences. These responses were assessed in mice exposed to low (0.25 Gy) or high (3 Gy) radiation doses at embryonic day 13.5 (E13.5), when NSCs giving rise to the cerebellum are specified but the external granule layer (EGL) has not yet formed, or at E16.5, during the expansion of granule PCs to form the EGL. We found crucial differences in DDR and apoptosis between NSCs and fate-restricted PCs, including lack of p21 expression in NSCs. NSCs also appear to be resistant to oncogenesis from low-dose radiation exposure but more vulnerable at higher doses. In addition, the pathway to DNA repair and the pattern of oncogenic alterations were strongly dependent on age at exposure, highlighting a differentiation-stage specificity of DNA repair pathways in NSCs and PCs. These findings shed light on the mechanisms used by NSCs and PCs to maintain genome integrity during neurogenesis and may have important implications for radiation risk assessment and for development of targeted therapies against brain tumors.

A long-held dogma in radiation biology has been that the biological effects of exposure to ionizing radiation occur as a result of damage in directly irradiated cells and that no effect would occur in neighboring unirradiated cells. This paradigm has been frequently challenged by reports of radiation effects in unirradiated or 'bystander' cells receiving signals from directly irradiated cells, an issue that may have substantial impact on radiation risk assessment and development of radiation-based therapies. Radiation-induced bystander effects have been shown in single-cell systems in vitro for an array of cancer relevant endpoints, and may trigger damage in more complex 3-D tissue systems. They may be mediated by soluble factors released by irradiated cells into the extracellular environment and/or by the passage of mediator molecules through gap-junction intercellular communication. To date, evidence that radiation-associated bystander or abscopal responses are effectual in vivo has been limited, but new data suggest that they may significantly affect tumor development in susceptible mouse models. Further understanding of how the signal/s is transmitted to unirradiated cells and tissues and how it provokes long-range and significant responses is crucial. By summarizing the existing evidence of radiation induced bystander-like effects in various systems with emphasis on in vivo findings, we will discuss the potential mechanisms involved in these observations and how effects in bystander cells contribute to uncertainties in assessing cancer risks associated with radiation exposure.

Ionizing radiation is a genotoxic agent and human carcinogen. Recent work has questioned long-held dogmas by showing that cancer-associated genetic alterations occur in cells and tissues not directly exposed to radiation, questioning the robustness of the current system of radiation risk assessment. In vitro, diverse mechanisms involving secreted soluble factors, gap junction intercellular communication (GJIC) and oxidative metabolism are proposed to mediate these indirect effects. In vivo, the mechanisms behind long-range 'bystander' responses remain largely unknown. Here, we investigate the role of GJIC in propagating radiation stress signals in vivo, and in mediating radiation-associated bystander tumorigenesis in mouse central nervous system using a mouse model in which intercellular communication is downregulated by targeted deletion of the connexin43 (Cx43) gene. We show that GJIC is critical for transmission of oncogenic radiation damage to the non-targeted cerebellum, and that a mechanism involving adenosine triphosphate release and upregulation of Cx43, the major GJIC constituent, regulates transduction of oncogenic damage to unirradiated tissues in vivo. Our data provide a novel hypothesis for transduction of distant bystander effects and suggest that the highly branched nervous system, similar to the vascular network, has an important role.

Heterozygous Patched1 (Ptc1(+/-)) mice are prone to medulloblastoma (MB), and exposure of newborn mice to ionizing radiation dramatically increases the frequency and shortens the latency of MB. In Ptc1(+/-) mice, MB is characterized by loss of the normal remaining Ptc1 allele, suggesting that genome rearrangements may be key events in MB development. Recent evidence indicates that brain tumors may be linked to defects in DNA-damage repair processes, as various combinations of targeted deletions in genes controlling cell-cycle checkpoints, apoptosis and DNA repair result in MB in mice. Non-homologous end joining (NHEJ) and homologous recombination (HR) contribute to genome stability, and deficiencies in either pathway predispose to genome rearrangements. To test the role of defective HR or NHEJ in tumorigenesis, control and irradiated Ptc1(+/-) mice with two, one or no functional Rad54 or DNA-protein kinase catalytic subunit (DNA-PKcs) alleles were monitored for MB development. We also examined the effect of Rad54 or DNA-PKcs deletion on the processing of endogenous and radiation-induced double-strand breaks (DSBs) in neural precursors of the developing cerebellum, the cells of origin of MB. We found that, although HR and NHEJ collaborate in protecting cells from DNA damage and apoptosis, they have opposite roles in MB tumorigenesis. In fact, although Rad54 deficiency increased both spontaneous and radiation-induced MB development, DNA-PKcs disruption suppressed MB tumorigenesis. Together, our data provide the first evidence that Rad54-mediated HR in vivo is important for suppressing tumorigenesis by maintaining genomic stability. Oncogene (2011) 30, 4740-4749; doi:10.1038/onc.2011.178; published online 23 May 2011