Simona Leonardi

Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum

The central dogma of radiation biology, that biological effects of ionizing radiation are a direct consequence of DNA damage occurring in irradiated cells, has been challenged by observations that genetic/epigenetic changes occur in unexposed “bystander cells” neighboring directly-hit cells, due to cell-to-cell communication or soluble factors released by irradiated cells. To date, the vast majority of these effects are described in cell-culture systems, while in vivo validation and assessment of biological consequences within an organism remain uncertain. Here, we describe the neonatal mouse cerebellum as an accurate in vivo model to detect, quantify, and mechanistically dissect radiation-bystander responses. DNA double-strand breaks and apoptotic cell death were induced in bystander cerebellum in vivo. Accompanying these genetic events, we report bystander-related tumor induction in cerebellum of radiosensitive Patched-1 (Ptch1) heterozygous mice after x-ray exposure of the remainder of the body. We further show that genetic damage is a critical component of in vivo oncogenic bystander responses, and provide evidence supporting the role of gap-junctional intercellular communication (GJIC) in transmission of bystander signals in the central nervous system (CNS). These results represent the first proof-of-principle that bystander effects are factual in vivo events with carcinogenic potential, and implicate the need for re-evaluation of approaches currently used to estimate radiation-associated health risks.

Role of connexin43 and ATP in long-range bystander radiation damage and oncogenesis in vivo

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.

Two-hit model for progression of medulloblastoma preneoplasia in Patched heterozygous mice

Inactivation of one Ptc1 allele predisposes humans and mice to spontaneous medulloblastoma development, and irradiation of newborn Ptc1 heterozygous mice results in dramatic increase of medulloblastoma incidence. While a role for loss of wild-type (wt) Ptc1 (LOH) in radiation-induced medulloblastomas from Ptc1neo67/+ mice is well established, the importance of this event in spontaneous medulloblastomas is still unclear. Here, we demonstrate that biallelic Ptc1 loss plays a crucial role in spontaneous medulloblastomas, as shown by high rate of wt Ptc1 loss in spontaneous tumors. In addition, remarkable differences in chromosomal events involving the Ptc1 locus in spontaneous and radiation-induced medulloblastomas suggest distinct mechanisms for Ptc1 loss. To assess when, during tumorigenesis, Ptc1 loss occurs, we characterized cerebellar abnormalities that precede tumor appearance in Ptc1neo67/+ mice. We show that inactivation of only one copy of Ptc1 is sufficient to give rise to abnormal cerebellar proliferations with different degree of altered cell morphology, but lacking potential to progress to neoplasia. Furthermore, we identify biallelic Ptc1 loss as the event causally related to the transition from the preneoplastic stage to full blown medulloblastoma. These results underscore the utility of the Ptc1neo67/+ mouse model for studies on the mechanisms of medulloblastoma and for development of new therapeutic strategies.

Hair Cycle–Dependent Basal Cell Carcinoma Tumorigenesis in Ptc1neo67/+ Mice Exposed to Radiation

We examined the effects of hair cycle phase on basal cell carcinoma (BCC) tumorigenesis induced by radiation in mice lacking one Patched allele (Ptc1(neo67/+)). Our results show that Ptc1(neo67/+) mouse skin irradiated in early anagen is highly susceptible to tumor induction, as a 3.2-fold incidence of visible BCC-like tumors was observed in anagen-irradiated compared with telogen-irradiated mice. Microscopic nodular BCC-like tumors were also enhanced by irradiation during active hair-follicle growth phases. Interestingly, histologic examination of the tumors revealed a qualitative difference in BCC tumorigenesis depending on hair growth phase at the time of exposure. In fact, in addition to typical BCC-like tumors, we observed development of a distinct basal cell tumor subtype characterized by anti-cytokeratin 14 and anti-smooth muscle actin reactivity. These tumors showed relatively short latency and rapid growth and were strictly dependent on age at irradiation, as they occurred only in mice irradiated in early anagen phase. Examination of anatomic and immunohistochemical relationships revealed a close relation of these tumors with the follicular outer root sheath of anagen skin. In contrast, there are strong indications for the derivation of typical, smooth muscle actin-negative BCC-like tumors from cell progenitors of interfollicular epidermis. These results underscore the role of follicular bulge stem cells and their progeny with high self-renewal capacity in the formation of basal cell tumors and contribute to clarify the relationship between target cell and tumor phenotype in BCC tumorigenesis induced by radiation.

Abscopal effect of radiation therapy: Interplay between radiation dose and p53 status

Abstract Purpose: This study investigates whether the abscopal effect induced by radiation-therapy (RT) is able to sterilize non-irradiated tumour cells through bystander signals. Material and methods: Wild-type (wt)-p53 or p53-null HCT116 human colon cancer cells were xenografted into both flanks of athymic female nude mice. When tumours reached a volume of 0.2 cm3, irradiation was performed, under strict dose monitoring, with a dedicated mobile accelerator designed for intra-Operative-RT (IORT). A dose of 10 or 20 Gy (IR groups), delivered by a 10 MeV electron beam, was delivered to a tumour established in one side flank, leaving the other non-irradiated (NIR groups). A subset of mice were sacrificed early on to carry out short-term molecular analyses. Results: All directly-irradiated tumours, showed a dose-dependent delayed and reduced regrowth, independent of the p53 status. Importantly, a significant effect on tumour-growth inhibition was also demonstrated in NIR wt-p53 tumours in the 20 Gy-irradiation group, with a moderate effect also evident after 10 Gy-irradiation. In contrast, no significant difference was observed in the NIR p53-null tumours, independent of the dose delivered. Molecular analyses indicate that p53-dependent signals might be responsible for the abscopal effect in our model system, via a pro-apoptotic pathway. Conclusions: We suggest that the interplay between delivered dose and p53 status might help to sterilize out-of-field tumour cells.

PARP-1 cooperates with Ptc1 to suppress medulloblastoma and basal cell carcinoma

The patched (Ptc1) protein is a negative regulator of sonic hedgehog signaling, a genetic pathway whose perturbation causes developmental defects and predisposition to specific malignant tumors. Humans and mice with mutated Ptc1 are prone to medulloblastoma and basal cell carcinoma (BCC), both tumors showing dependence on radiation damage for rapid onset and high penetrance. Poly(ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. In healthy and fertile PARP-1-null mice, radiation exposure reveals an extreme sensitivity and a high genomic instability. To test for interactions between PARP-1 and sonic hedgehog signaling, PARP-1-null mice were crossed to Ptc1 heterozygous mice. PARP-1 deletion further accelerated medulloblastoma development in irradiated Ptc1(+/-) mice, showing that PARP-1 inactivation sensitizes cerebellar cells to radiation tumorigenic effects. In addition to increased formation and slowed down kinetics of disappearance of gamma-H2AX foci, we observed increased apoptosis in PARP-1-deficient granule cell progenitors after irradiation. Double-mutant mice were also strikingly more susceptible to BCC, with >50% of animals developing multiple, large, infiltrative tumors within 30 weeks of age. The results provide genetic evidence that PARP-1 function suppresses sonic hedgehog pathway-associated tumors arising in response to environmental stress.

Physical, Heritable and Age-Related Factors as Modifiers of Radiation Cancer Risk in Patched Heterozygous Mice

PURPOSE To address the tumorigenic potential of exposure to low/intermediate doses of ionizing radiation and to identify biological factors influencing tumor response in a mouse model highly susceptible to radiogenic cancer. METHODS AND MATERIALS Newborn Ptc1 heterozygous mice were exposed to X-ray doses of 100, 250, and 500 mGy, and tumor development was monitored for their lifetime. Additional groups were irradiated with the same doses and sacrificed at fixed times for determination of short-term endpoints, such as apoptosis and early preneoplastic lesions in cerebellum. Finally, groups of Ptc1 heterozygous mice were bred on the C57BL/6 background to study the influence of common variant genes on radiation response. RESULTS We have identified a significant effect of low-intermediate doses of radiation (250 and 500 mGy) in shortening mean survival and inducing early and more progressed stages of tumor development in the cerebellum of Ptc1(+/-) mice. In addition, we show that age at exposure and heritable factors are potent modifiers of radiation-related cancer risk. CONCLUSIONS The Ptc1 knockout mouse model offers a highly sensitive system that may potentially help to improve understanding and quantification of risk at low doses, such as doses experienced in occupational and medical exposures, and clarify the complex interactions between genetic and environmental factors underlying cancer susceptibility.

Developmental and oncogenic radiation effects on neural stem cells and their differentiating progeny in mouse cerebellum

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. Stem Cells 2013;31:2506–2516

Oncogenic Radiation Abscopal Effects In Vivo: Interrogating Mouse Skin

PURPOSE To investigate the tissue dependence in transmission of abscopal radiation signals and their oncogenic consequences in a radiosensitive mouse model and to explore the involvement of gap junction intercellular communication (GJIC) in mediating radiation tumorigenesis in off-target mouse skin. METHODS AND MATERIALS Patched1 heterozygous (Ptch1(+/-)) mice were irradiated at postnatal day 2 (P2) with 10 Gy of x-rays. Individual lead cylinders were used to protect the anterior two-thirds of the body, whereas the hindmost part was directly exposed to radiation. To test the role of GJICs and their major constituent connexin43 (Cx43), crosses between Ptch1(+/-) and Cx43(+/-) mice were similarly irradiated. These mouse groups were monitored for their lifetime, and skin basal cell carcinomas (BCCs) were counted and recorded. Early responses to DNA damage - Double Strand Breaks (DSBs) and apoptosis - were also evaluated in shielded and directly irradiated skin areas. RESULTS We report abscopal tumor induction in the shielded skin of Ptch1(+/-) mice after partial-body irradiation. Endpoints were induction of early nodular BCC-like tumors and macroscopic infiltrative BCCs. Abscopal tumorigenesis was significantly modulated by Cx43 status, namely, Cx43 reduction was associated with decreased levels of DNA damage and oncogenesis in out-of-field skin, suggesting a key role of GJIC in transmission of oncogenic radiation signals to unhit skin. CONCLUSIONS Our results further characterize the nature of abscopal responses and the implications they have on pathologic processes in different tissues, including their possible underlying mechanistic bases.

Developmental and oncogenic effects of Insulin-like Growth Factor-I in Ptc1+/- mouse cerebellum

BackgroundMedulloblastoma is amongst the most common malignant brain tumors in childhood, arising from neoplastic transformation of granule neuron precursors (GNPs) of the cerebellum via deregulation of pathways involved in cerebellar development. Deregulation of the Sonic hedgehog/Patched1 (Shh/Ptc1) signaling pathway predisposes humans and mice to medulloblastoma. In the brain, insulin-like growth factor (IGF-I) plays a critical role during development as a neurotrophic and neuroprotective factor, and in tumorigenesis, as IGF-I receptor is often activated in medulloblastomas.ResultsTo investigate the mechanisms of genetic interactions between Shh and IGF signaling in the cerebellum, we crossed nestin/IGF-I transgenic (IGF-I Tg) mice, in which transgene expression occurs in neuron precursors, with Ptc1+/-knockout mice, a model of medulloblastoma in which cancer develops in a multistage process. The IGF-I transgene produced a marked brain overgrowth, and significantly accelerated tumor development, increasing the frequency of pre-neoplastic lesions as well as full medulloblastomas in Ptc1+/-/IGF-I Tg mice. Mechanistically, tumor promotion by IGF-I mainly affected preneoplastic stages through de novo formation of lesions, while not influencing progression rate to full tumors. We also identified a marked increase in survival and proliferation, and a strong suppression of differentiation in neural precursors.ConclusionsAs a whole, our findings indicate that IGF-I overexpression in neural precursors leads to brain overgrowth and fosters external granular layer (EGL) proliferative lesions through a mechanism favoring proliferation over terminal differentiation, acting as a landscape for tumor growth. Understanding the molecular events responsible for cerebellum development and their alterations in tumorigenesis is critical for the identification of potential therapeutic targets.