Activating transcription factor 4 (ATF4) is a transcription factor induced under severe hypoxia and a component of the PERK pathway involved in the unfolded protein response (UPR), a process that protects cells from the negative consequences of endoplasmic reticulum (ER) stress. In this study, we have used small interfering RNA (siRNA) and microarray analysis to provide the first whole-genome analysis of genes regulated by ATF4 in cancer cells in response to severe and prolonged hypoxic stress. We show that ATF4 is required for ER stress and hypoxia-induced expansion of autophagy. MAP1LC3B (LC3B) is a key component of the autophagosomal membrane, and in this study we demonstrate that ATF4 facilitates autophagy through direct binding to a cyclic AMP response element binding site in the LC3B promoter, resulting in LC3B upregulation. Previously, we have shown that Bortezomib-induced ATF4 stabilization, which then upregulated LC3B expression and had a critical role in activating autophagy, protecting cells from Bortezomib-induced cell death. We also showed that severe hypoxia stabilizes ATF4. In this study, we demonstrate that severe hypoxia leads to ER stress and induces ATF4-dependent autophagy through LC3 as a survival mechanism. In summary, we show that ATF4 has a key role in the regulation of autophagy in response to ER stress and provide a direct mechanistic link between the UPR and the autophagic machinery.

The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G 2 phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxiainduced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/ 53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.Sensing and responding to DNA damage is crucial for maintaining cellular homeostasis and preventing the development of cancer. The cellular response to DNA damage can be divided into three parts: sensing the type of damage, activating DNA damage signaling pathways, and repairing the damage. The proteins involved in these three processes act as sensors, transducers, and effectors of the DNA damage response (41, 54). The ATM kinase is one of the key transducers of the DNA double-stranded break response. ATM has been identified as the product mutated or inactivated in ataxia telangiectasia (AT) patients and belongs to the phosphatidyl inositol-3-kinase-like kinase (PIKK) family, together with its family members ATR, DNA-PK, and mTOR. Elegant studies have demonstrated that ATM is present as an inactive dimer that undergoes rapid autophosphorylation on serine 1981 after DNA damage (4) and is recruited to sites of DNA strand breaks (2). Various proteins, such as the MRE11-Rad50-NBS1 (MRN) complex (49) and MDC1 (20, 43), have been described to be essential for the efficient response of ATM to DNA damage. Activated ATM phosphorylates downstream targets, such as p53, Chk2, and BRCA 1 and 2, that are involved in DNA repair, cell cycle control, and apoptosis.Low-oxygen tension or hypoxia is a common feature in all solid tumors (15). It is strongly associated with tumor development, malignant progression, metastatic outgrowth, and resistance to therapy and is considered an independent prognostic indicator for poor patient prognosis in various tumor types. Tumor hypoxia results from an imbalance between the cellular oxygen consumption rate of cells and the delivery of oxygen to cells (50). Interestingly, the level of tumor hy...