ATM Activation In Response Research Articles

Mechanisms of Non-canonical Activation of Ataxia Telangiectasia Mutated.

ATM is a master regulator of the cellular response to DNA damage. The classical mechanism of ATM activation involves its monomerization in response to DNA double-strand breaks, resulting in ATM-dependent phosphorylation of more than a thousand substrates required for cell cycle progression, DNA repair, and apoptosis. Here, new experimental evidence for non-canonical mechanisms of ATM activation in response to stimuli distinct from DNA double-strand breaks is discussed. It includes cytoskeletal changes, chromatin modifications, RNA-DNA hybrids, and DNA single-strand breaks. Noncanonical ATM activation may be important for the pathology of the multisystemic disease Ataxia Telangiectasia.

Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1.

Hypoxia-induced replication stress is one of the most physiologically relevant signals known to activate ATM in tumors. Recently, the ATM interactor (ATMIN) was identified as critical for replication stress-induced activation of ATM in response to aphidicolin and hydroxyurea. This suggests an essential role for ATMIN in ATM regulation during hypoxia, which induces replication stress. However, ATMIN also has a role in base excision repair, a process that has been demonstrated to be repressed and less efficient in hypoxic conditions. Here, we demonstrate that ATMIN is dispensable for ATM activation in hypoxia and in contrast to ATM, does not affect cell survival and radiosensitivity in hypoxia. Instead, we show that in hypoxic conditions ATMIN expression is repressed. Repression of ATMIN in hypoxia is mediated by both p53 and HIF-1α in an oxygen dependent manner. The biological consequence of ATMIN repression in hypoxia is decreased expression of the target gene, DYNLL1. An expression signature associated with p53 activity was negatively correlated with DYNLL1 expression in patient samples further supporting the p53 dependent repression of DYNLL1. Together, these data demonstrate multiple mechanisms of ATMIN repression in hypoxia with consequences including impaired BER and down regulation of the ATMIN transcriptional target, DYNLL1.

Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis.

Oxidative stress shapes breast cancer phenotype through chronic activation of ATM-dependent signaling.

Reactive oxygen species (ROS) are thought to be among the initiating insults that drive carcinogenesis; however, beyond the mutagenic properties of ROS, it is unclear how reactive oxygen species and response to redox imbalance may shape cancer phenotype. We have previously observed that basal activity of the powerfully oncogenic transcription factor NF-κB in cultured breast cancer and other tumor cell lines is dependent upon the DNA damage-responsive kinase ATM. Here we show that, in MDA-MB-231 and HeLa cells, basal ATM-dependent NF-κB activation occurs through a canonical DNA damage-responsive signaling pathway as knockdown of two proteins involved in this signaling pathway, ERC1 and TAB1, results in loss of NF-κB basal activity. We further show that knockdown of ATM in MDA-MB-231, a breast cancer line with a pronounced mesenchymal phenotype, results in the reversion of these cells to an epithelial morphology and gene expression pattern. Culture of MDA-MB-231 and HeLa cells on the antioxidant N-acetyl cysteine (NAC) blunted NF-κB transcriptional activity, and long-term culture on low doses of NAC resulted in coordinate reductions in steady-state ROS levels, acquisition of an epithelial morphology, as well as upregulation of epithelial and downregulation of mesenchymal marker gene expression. Moreover, these reversible effects are attributable, at least in part, to downregulation of ATM-dependent NF-κB signaling in MDA-MB-231 cells as RNAi-mediated knockdown of the NF-κB subunit RelA or its upstream activator TG2 produced similar alterations in phenotype. We conclude that chronic activation of ATM in response to persistent ROS insult triggers continual activation of the oncogenic NF-κB transcriptional complex that, in turn, promotes aggressive breast cancer phenotype.

Abstract A92: The role of FOXM1 and NBS1 in DNA damage-induced senescence and epirubicin resistance.

Abstract Background: The Forkhead box M1 (FOXM1) transcription factor is responsible for the regulation of a wide range of biological processes, including cell cycle, cell proliferation, apoptosis and tumorigenesis. Deregulation of FOXM1 has been suggested to be associated with drug resistance and poor prognosis in breast cancer patients and this could occur through an enhancement of DNA damage repair as well as a reduced propensity to undergo cell death or senescence. However, the exact mechanisms of how FOXM1 modulates drug resistance still remain unclear. The aim of this study is to investigate the new roles of FOXM1 and its downstream targets in DNA damage response and cellular senescence, which are vital for both the tumor progression and the development of chemotherapeutic drug resistance. Materials and Methods: MCF-7 human breast carcinoma cell lines and mouse embryonic fibroblasts (MEFs) were exposed to epirubicin and long-term cell viability was assessed by clonogenic assay. DNA double strand break repair was examined by γH2AX foci and DR-GFP repair assay. Analysis of cellular senescence was based on cellular β-galactosidase activity and changes in cell morphology. Expression levels of key DNA repair genes were assessed by using quantitative real-time PCR and western blot analysis. Results: We previously found that FOXM1 is required for DNA repair via homologous recombination repair. Consistently, reconstitution of FOXM1 in FOXM1-deficient MEFs decreased the accumulation of senescence-associated γH2AX foci after epirubicin treatment. Here, we shows that FOXM1-depletion can sensitize both human breast cancer MCF-7 cells and MEFs into entering epirubicin-induced senescence as indicated by the loss of long-term cell proliferation ability, the accumulation of persistent γH2AX foci, and the induction of senescence-associated β-galactosidase activity. Interestingly, we also demonstrate that FOXM1 regulates NBS1, a key component for the MRN complex-mediated ATM activation in response to DNA double stranded break (DSBs), at the transcriptional level. Using HeLa cell lines harbouring the DR-GFP reporter, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, suggesting that NBS1 is indispensable for the HR function of FOXM1. Similar to FOXM1, NBS1 is strongly up-regulated in the epirubicin-resistant MCF-7 (MCF-7 EpiR) cells compared with parental MCF-7 cells and the depletion of FOXM1 or NBS1 also renders MCF-7 and MCF-7 EpiR cells more sensitive to epirubicin-induced cellular senescence. Conclusion: Taken together, these results indicate that NBS1 is a key FOXM1 downstream target gene involved in DNA damage response, DNA damage-induced senescence and epirubicin resistance. The FOXM1-NBS1 axis can be a reliable prognostic marker and a potential therapeutic target for overcoming genotoxic agent resistance in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A92. Citation Format: Pasarat Khongkow, Upekha Karunarathna, Eric Lam. The role of FOXM1 and NBS1 in DNA damage-induced senescence and epirubicin resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A92.

NBS1 directly activates ATR independently of MRE11 and TOPBP1

NBS1 plays unique and essential roles in ATM activation in response to DNA double-strand breaks. We found that CHK1 phosphorylation and FANCD2 ubiquitination induced by various DNA replication-stalling agents were abrogated in Nbs1 knockout DT40 cells but not in conditional Mre11 knockout cells, indicating an MRE11-independent role for NBS1 in ATR activation. The results of in vitro ATR kinase assay indicated that the N-terminal region of NBS1 directly activates ATR independently of TOPBP1, consistent with the findings that this region of NBS1 directly interacts with ATR. This conclusion was furthermore supported by the results of in vivo experiments; the expression of the N-terminal region of NBS1 fused to PCNA induces ATR activation in Rad17 knockout cells, and the expression of the ATR activation domain of TOPBP1 fused to PCNA induces ATR activation in Nbs1 knockout cells. These results therefore indicate that NBS1 and TOPBP1 have the potential to activate ATR independently, although both are required for functional activation of ATR in vivo.

Skp2 E3 Ligase Integrates ATM Activation and Homologous Recombination Repair by Ubiquitinating NBS1

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.

Regulator of G protein Signaling 6 (RGS6) is Upregulated in Cancer cells by Doxorubicin and Modulates Doxorubicin‐induced DNA damage signaling

RGS proteins were discovered as negative regulators of heterotrimeric G protein signaling. The precise biological role of all but a few RGS proteins is unknown, as is the need for such a large structurally diverse protein family if RGS proteins function only as G protein GAPs. Here, we provide new evidence suggesting a role of RGS6 in regulating DNA damage responses in cancer cells. Doxorubicin (Dox) treatment of cancer cell lines led to dramatic increases in RGS6 protein levels, which were undetectable in unstimulated cells. Dox induction of RGS6 protein was accompanied by significant increase in RGS6 gene transcription as assessed using an RGS6 gene promoter reporter. Further, expression of RGS6 in MCF7 cells promoted phosphorylation of p53 in response to a low dose of Dox, which alone had no effects. This activity of RGS6 requires its DEP domain but is independent of its interaction with G proteins. RGS6 expression in several cancer cell lines promoted cell cycle arrest and growth suppression. ATM, an upstream activator of p53, may be involved in RGS6‐induced p53 phosphorylation, as expression of RGS6 promotes ATM activation in response to a submaximal dose of Dox. Our data identify RGS6 as a target of Dox and suggest that RGS6 may play a novel role as a modulator of chemosensitivity in cancer cells. Importantly, these actions of RGS6 are independent of its interaction with G proteins.NIH (GM075033) and AHA (0750057Z).

Males absent on the first (MOF): from flies to humans

Histone modifications such as acetylation, methylation and phosphorylation have been implicated in fundamental cellular processes such as epigenetic regulation of gene expression, organization of chromatin structure, chromosome segregation, DNA replication and DNA repair. Males absent on the first (MOF) is responsible for acetylating histone H4 at lysine 16 (H4K16) and is a key component of the MSL complex required for dosage compensation in Drosophila. The human ortholog of MOF (hMOF) has the same substrate specificity and recent purification of the human and Drosophila MOF complexes showed that these complexes were also highly conserved through evolution. Several studies have shown that loss of hMOF in mammalian cells leads to a number of different phenotypes; a G2/M cell cycle arrest, nuclear morphological defects, spontaneous chromosomal aberrations, reduced transcription of certain genes and an impaired DNA repair response upon ionizing irradiation. Moreover, hMOF is involved in ATM activation in response to DNA damage and acetylation of p53 by hMOF influences the cell's decision to undergo apoptosis instead of a cell cycle arrest. These data, highlighting hMOF as an important component of many cellular processes, as well as links between hMOF and cancer will be discussed.

Nbs1 moving up in the world

The Nbs1 protein participates in the cell-cycle checkpoint response to DNA double-strand breaks (DSBs), but its precise mode of action — especially in relation to the checkpoint kinase Atm — has been debated. New mouse models suggest that Nbs1 is required for activation of Atm in response to DNA DSBs and that it also functions in an amplification loop with Atm that allows even a small number of DNA DSBs to mount a potent checkpoint response.

ATM Activation and Its Recruitment to Damaged DNA Require Binding to the C Terminus of Nbs1

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.

ATM Is Activated in Response toN-Methyl-N′-nitro- N-nitrosoguanidine-induced DNA Alkylation

p53 plays an important role in response to ionizing radiation by regulating cell cycle progression and triggering apoptosis. These activities are controlled, in part, by the phosphorylation of p53 by the protein kinase ATM. Recent evidence indicates that the monofunctional DNA alkylating agent N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) also triggers up-regulation and phosphorylation of p53; however, the mechanism(s) responsible for this are unknown. We observed that in MNNG-treated normal human fibroblasts, up-regulation and phosphorylation of p53 was sensitive to the ATM kinase inhibitor wortmannin. ATM-deficient fibroblasts exhibited a delay in p53 up-regulation indicating a role for ATM in triggering the MNNG-induced response. Likewise, a mismatch repair (MMR)-deficient colorectal tumor line failed to show rapid up-regulation of p53. However, unlike ATM-deficient cells, these MMR-deficient cells displayed rapid phosphorylation of the p53 residue serine 15 after MNNG. In vitro kinase assays indicate that ATM is rapidly activated in both normal and MMR-deficient cells in response to MNNG. Using a number of morphological and biochemical approaches, we failed to observe MNNG-induced apoptosis in normal human fibroblasts, suggesting that apoptosis-induced DNA strand breaks are not required for the activation of ATM in response to MNNG. Comet assays indicated that strand breaks accumulated, and p53 up-regulation/phosphorylation occurred quite rapidly (within 30 min) after MNNG treatment, suggesting that DNA strand breaks that arise during the repair process activate ATM. These findings indicate that ATM activation is not limited to the ionizing radiation-induced response and potentially plays an important role in response to DNA alkylation.