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

Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of a lung tumor xenograft

Poly(ADP-ribose) polymerase-1 is a critical enzyme in the repair of DNA strand breaks. Inhibition of PARP-1 increases the effectiveness of radiation in killing tumor cells. However, while the mechanism(s) are well understood for these radiosensitizing effects in vitro, the underlying mechanism(s) in vivo are less clear. Nicotinamide, a drug structurally related to the first generation PARP-1 inhibitor, 3-aminobenzamide, reduces tumor hypoxia by preventing transient cessations in tumor blood flow, thus improving tumor oxygenation and sensitivity to radiotherapy. Here we investigate whether olaparib, a potent PARP-1 inhibitor, enhances radiotherapy, not only by inhibiting DNA repair but also by changing tumor vascular haemodynamics in non-small cell lung carcinoma. In irradiated Calu-6 and A549 cells, olaparib enhanced the cytotoxic effects of radiation (SER(10)=1.5 and 1.3) and DNA double strand breaks persisted for at least 24 h after treatment. Combination treatment of Calu-6 xenografts with olaparib and fractionated radiotherapy caused significant tumor regression (p=0.007) relative to radiotherapy alone. To determine whether this radiosensitisation was due solely to effects on DNA repair we used a dorsal window chamber model to establish the drug/radiation effects on vessel dynamics. Olaparib alone, when given as single or multiple daily doses, or in combination with fractionated radiotherapy, increased the perfusion of tumor blood vessels. Furthermore, an ex vivo assay in phenylephrine pre-constricted arteries confirmed olaparib to have higher vasodilatory properties than nicotinamide. This study suggests that olaparib warrants consideration for further development in combination with radiotherapy in clinical oncology settings such as NSCLC

The role of the HIF-1α transcription factor in increased cell division at physiological oxygen tensions

HIF-1 is a transcription factor that mediates the cellular responses to low oxygen environments, mainly as a result of having an oxygen-labile subunit, HIF-1α. HIF-1α has been carefully studied in the context of severe hypoxic stresses (<1% O[subscript 2]), but it is also known to be present at oxygen tensions commonly found in normal tissues in vivo (∼1-13% O[subscript 2]), albeit at much lower levels. Its role under these physiological conditions is not fully understood. Here, we show that a transcriptionally active HIF-1α was up-regulated at 5% O[subscript 2], both in normal and cancer cells, but only some of its target genes were elevated as a result. HIF-1α induction was in part dependent on the activation of the ERK1/2 MAPK signalling pathway, which we have previously shown is active at 5% O[subscript 2]. We also found that HIF-1α does not contribute to the protection against DNA damage that can be observed in low oxygen environments, and that there are certain DNA damaging agents, such as doxorubicin and actinomycin D, that prevent HIF-1α induction independently of p53. Moreover, absence of HIF-1α significantly reduced the growth advantage of cells cultured at 5% O[subscript 2]. In view of these data, we conclude that HIF-1α can be induced and activated at physiological oxygen tensions in a MAPK-dependent manner and that, although this does not lead to pro-survival responses to stress, it determines the increased cell proliferation rates that are common under these conditions

Inhibition of HIF-1α expression by DNA damaging agents.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells treated with 1.25 µM U0126 and 0.4 µg/ml doxorubicin, and cultured at 20% or 5% O₂ for 2 days. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α in HCT116 and HCT116 p53^−/− in lysates collected 24 hours after treatment with 1 µg/ml Actinomicyn D (ActD) or 200 µM tert-Butyl Hydroperoxyde (tBH) for 2hours (<b>C</b>) Western blot showing the protein levels of HIF-1α, p53 and p21 in HCT116 and MCF-7 cells treated with 0.4 µg/ml doxorubicin or 10 Gy γ-radiation for 24 hours at 20% or <1% O₂ (hypoxia).</p

HIF-1α has no effect on the activation of MAPK at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, p53, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells transfected with 200 pmol of siRNA against HIF-1α and treated with doxorubicin (0.4 µg/ml). Control cells were transfected with a luciferase siRNA instead. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 treated with 40 µM YC-1 and/or 0.4 µg/ml doxorubicin and cultured at 20% or 5% O₂ for 24 hours. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>C</b>) Representative FACS plots of HCT116 cells stained with PI. Cells were transfected with 50 pmol of siRNA against HIF-1α and treated with 0.4 µg/ml doxorubicin. Cells were cultured at 20% or 5% O₂ for 24 hours. Percentages indicate number of subG₁ events (dead cells).</p

Physiological oxygen tensions induce HIF-1α expression and activity.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116. Cells were cultured at 20% or 5% O₂ for 72 hours. (<b>B</b>) Western blot showing the protein levels of HIF-1α in normal human keratinocytes cultured for 1 to 4 days at 5% O₂ or treated 500 µM of chemical hypoximimetic CoCl₂ for 16 hours (H). (<b>C</b>) Luciferase assay showing HIF-1α in HCT116 cells. HCT116 were transfected with a PGK-1 luciferase reporter plasmid and a β-galactosidase control plasmid and then cultured for 48 hours at 5% O₂. β-galactosidase activity was used to normalize luciferase activity. Luciferase activity is expressed as a ratio to 20% O₂ levels. Results show mean values of 3 independent experiments and error bars represent standard deviation (<b>D</b>) qRT-PCR showing mRNA levels of Glut-1, PGK-1 and VEGF in HCT116 cells cultured at 20% and 5% O₂ for 24 hours. Results show mean values of 3 independent experiments and error bars represent standard deviation. P values (unpaired t-tests): 0.03 (*), 0.001 (**), 0.7 (ns). (E) Western blot of lysates of HCT116 cultured at 20% or 5% O₂ for 72 hours, showing expression of Glut-1 and PHD2.</p

HIF-1α contributes to increased proliferation of cells at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116 HIF^+/+ and HIF^−/−cultured either at 20% O₂ or under hypoxic stress (<0.1% O₂) for 16 hours. (<b>B</b>) Proliferation curves of HIF^+/+ and HIF^−/− HCT116 cells cultured at 20% or 5% O₂ from 2 to 8 days. Values represent ratio of cell numbers normalized to the initial seeded cells (10⁶). (<b>C</b>) Representative colony formation assay for HCT116 HIF^+/+ and HIF^−/−cultured at 20% or 5% O₂. 200 cells were seeded in each plate and 14 days later they were stained with Giemsa. Media was not changed during the process. (<b>D</b>) Percentage of EdU positive HCT116 HIF^+/+ and HIF^−/− cells as assessed by immunofluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097938#pone.0097938.s002" target="_blank">Figure S2</a>). Cells were incubated with EdU for 30 minutes in the corresponding oxygen tensions. Results represent means of two independent experiments. Two microscope fields were scored in each experiment. Error bars represent standard error. P value (unpaired t-test): 0.0127 (*), (<b>E</b>) Proposed model of the roles of HIF-1 at different oxygen concentrations.</p

Chemical inhibition of MAPK reduces the activation of HIF-1α.

<p>Western blot showing the protein levels of HIF-1α and phosphorylated (active) ERK 1/2 MAPK in HCT116 cultured at 20% or 5% O₂for 12 to 48 hours, in the presence of 1.25 µM U0126. U0126 was added at the same time cells were transferred to 5%O₂. Total MAPK levels are provided as loading control.</p