Replication stress and chromatin context link ATM activation to a role in DNA replication

ATM-mediated signaling in response to DNA damage is a barrier to tumorigenesis. Here we asked whether replication stress could also contribute to ATM signaling. We demonstrate that, in the absence of DNA damage, ATM responds to replication stress in a hypoxia-induced heterochromatin-like context. In certain hypoxic conditions, replication stress occurs in the absence of detectable DNA damage. Hypoxia also induces H3K9me3, a histone modification associated with gene repression and heterochromatin. Hypoxia-induced replication stress together with increased H3K9me3 leads to ATM activation. Importantly, ATM prevents the accumulation of DNA damage in hypoxia. Most significantly, we describe a stress-specific role for ATM in maintaining DNA replication rates in a background of increased H3K9me3. Furthermore, the ATM-mediated response to oncogene-induced replication stress is enhanced in hypoxic conditions. Together, these data indicate that hypoxia plays a critical role in the activation of the DNA damage response, therefore contributing to this barrier to tumorigenesis

HIF-1 alpha-independent hypoxia-induced rapid PTK6 stabilization is associated with increased motility and invasion

© 2014 Landes Bioscience. PTK6/Brk is a non-receptor tyrosine kinase overexpressed in cancer. Here we demonstrate that cytosolic PTK6 is rapidly and robustly induced in response to hypoxic conditions in a HIF-1-independent manner. Furthermore, a proportion of hypoxic PTK6 subsequently re-localized to the cell membrane. We observed that the rapid stabilization of PTK6 is associated with a decrease in PTK6 ubiquitylation and we have identified c-Cbl as a putative PTK6 E3 ligase in normoxia. The consequences of hypoxia-induced PTK6 stabilization and subcellular re-localization to the plasma membrane include increased cell motility and invasion, suggesting PTK6 targeting as a therapeutic approach to reduce hypoxia-regulated metastatic potential. This could have particular significance for breast cancer patients with triple negative disease

Effects of acute versus chronic hypoxia on DNA damage responses and genomic instability.

Questions exist concerning the effects of acute versus chronic hypoxic conditions on DNA replication and genomic stability that may influence tumorigenesis. Severe hypoxia causes replication arrest independent of S-phase checkpoint, DNA damage response, or transformation status. Arrests occur during both the initiation and elongation phases of DNA replication, correlated with a rapid decrease in available deoxynucleotide triphosphates. With fluctuating oxygen tensions in tumors, arrested hypoxic cells may undergo rapid reperfusion and reoxygenation that leads to reoxygenation-induced DNA damage. In cells subjected to chronic hypoxia, we found that replicative restart was inhibited along with numerous replication factors, including MCM6 and RPA, the latter of which limits the hypoxia-induced DNA damage response. In contrast, in cells where replicative restart occurred, it was accompanied by extensive reoxygenation-induced DNA damage and compromised DNA repair. We found that cells reoxygenated after acute hypoxia underwent rapid p53-dependent apoptosis. Our findings suggest that cells lacking functional p53 are more susceptible to genomic instability and potentially tumorigenesis if they experience reoxygenation after acute exposure to hypoxia

CH-01 is a hypoxia-activated prodrug that sensitizes cells to hypoxia/reoxygenation through inhibition of Chk1 and aurora A

The increased resistance of hypoxic cells to all forms of cancer therapy presents a major barrier to the successful treatment of most solid tumors. Inhibition of the essential kinase Checkpoint kinase 1 (Chk1) has been described as a promising cancer therapy for tumors with high levels of hypoxia-induced replication stress. However, as inhibition of Chk1 affects normal replication and induces DNA damage, these agents also have the potential to induce genomic instability and contribute to tumorigenesis. To overcome this problem, we have developed a bioreductive prodrug, which functions as a Chk1/Aurora A inhibitor specifically in hypoxic conditions. To achieve this activity, a key functionality on the Chk1 inhibitor (CH-01) is masked by a bioreductive group, rendering the compound inactive as a Chk1/Aurora A inhibitor. Reduction of the bioreductive group nitro moiety, under hypoxic conditions, reveals an electron-donating substituent that leads to fragmentation of the molecule, affording the active inhibitor. Most importantly, we show a significant loss of viability in cancer cell lines exposed to hypoxia in the presence of CH-01. This novel approach targets the most aggressive and therapy-resistant tumor fraction while protecting normal tissue from therapy-induced genomic instability. © 2013 American Chemical Society

SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

Hypoxia is associated with tumor radioresistance; therefore, a predictive marker for tumor hypoxia and a rational target to overcome it have been sought to realize personalized radiotherapy. Here, we show that serine protease inhibitor Kazal type I (SPINK1) meets these 2 criteria. SPINK1 expression was induced upon hypoxia (O2 < 0.1%) at the transcription initiation level in a HIF-dependent manner, causing an increase in secreted SPINK1 levels. SPINK1 proteins were detected both within and around hypoxic regions of xenografted and clinical tumor tissues, and their plasma levels increased in response to decreased oxygen supply to xenografts. Secreted SPINK1 proteins enhanced radioresistance of cancer cells even under normoxic conditions in EGFR-dependent and nuclear factor erythroid 2–related factor 2–dependent (Nrf2-dependent) manners and accelerated tumor growth after radiotherapy. An anti-SPINK1 neutralizing antibody exhibited a radiosensitizing effect. These results suggest that SPINK1 secreted from hypoxic cells protects the surrounding and relatively oxygenated cancer cells from radiation in a paracrine manner, justifying the use of SPINK1 as a target for radiosensitization and a plasma marker for predicting tumor hypoxia

Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double strand breaks (DSB) which co-localized with 'replication factories' and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (&gt;1Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro. These data identify RS as a cancer stem cell-specific target with significant clinical potential

Human AlkB Homologue 5 Is a Nuclear 2-Oxoglutarate Dependent Oxygenase and a Direct Target of Hypoxia-Inducible Factor 1α (HIF-1α)

Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia

Hypoxia-induced SETX links replication stress with the unfolded protein response.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways

Alpha-particle-induced complex chromosome exchanges transmitted through extra-thymic lymphopoiesis in vitro show evidence of emerging genomic instability

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.This work was supported by the Department of Health, UK. Contract RRX95 (RMA NSDTG)

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

[Background] The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded.[Methods] In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes.[Results] In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction.[Conclusions] These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.This work was supported by Junta de Andalucía, project of Excellence from Junta de Andalucía P10-CTS-0662, P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01, SAF2015-70520- R, RTI2018-098968-B-I00, RTICC RD12/0036/0026 and CIBER Cáncer ISCIII CB16/12/00421 to FJO. EB1s lab is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the MINECO (CB16/12/00421) grants. Fundación Domingo Martínez (call 2019).Peer reviewe