Multiple pathways are involved in the anoxia response of SKIP3 including HuR-regulated RNA stability, NF-κB and ATF4

Abstract

Under anoxia a coordinated, cytoprotective program is induced, called the unfolded protein response (UPR). Activating transcription factor 4 (ATF4) is a mediator of the UPR and activates a gene expression program, promoting tumour growth and survival under anoxia. A key gene induced by ATF4 under normoxic conditions is SKIP3. We characterized the induction of SKIP3 during anoxic exposure to determine whether UPR alone was sufficient or there was a more complex regulatory response to anoxia. There was temporal separation of acute hypoxia-inducible factor (HIF)-1a-and chronic ATF4-dependent gene expression programs. SKIP3 was regulated by chronic (48 h) rather than acute anoxia (o24 h) by a complex set of pathways and mechanisms, besides ATF4 induced by the classical UPR, there was transcriptional regulation by nuclear factor-kappa B (NFjB) and RNA stabilization by HuR. Temporal activation of the NF-jB pathway under anoxia protected cells from negative consequences of the oxygen stress and involved the canonical signalling pathways that promote IjBA phosphorylation and degradation, and reduced mRNA level of the inhibitory protein IjBA followed by the translational repression of IjBA. We also show that SKIP3 acts as an inhibitor of NF-jB and ATF4-dependent transcription under anoxia and provides a regulatory feedback loop. Repression of the survival pathway NF-jB by SKIP3 sensitized cells to metabolic consequences of the anoxic stress. Thus, the response to anoxia is mediated by three pathways independently of HIF, suggesting that combined therapeutic approaches would be needed to maximize effects against this pathway.

Regulation of autophagy by ATF4 in response to severe hypoxia

“…In real-time quantitative PCR experiments, the methods used by us for extraction, quantification and evaluation of the quality of RNA were as previously described (Rzymski et al, 2008), the data of real-time quantitative PCR were normalized to tubulin alpha 6 and b-actin (for the list of primers, see Supplementary Materials).…”

“…Activating transcription factor 4 regulates the integrated stress response, a gene expression program involved in oxidative stress, amino acid synthesis, differentiation, metastasis, angiogenesis (Harding et al, 2003) and drug resistance . Acute HIF1a and chronic ATF4-dependent gene expression programs are temporarily separated in cancer cells during exposure to hypoxia (Rzymski et al, 2008).…”

Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

“…This same anoxia pathway can also be activated by nutritional deficiencies such as glucose and/or amino acid deprivation, and can lead to ERSR and translational increase of ATF4 (Harding et al, 2000). In addition to an increase in mRNA translation of factors related to the unfolded protein response pathway, such as ATF4, cancer cells also respond to anoxia via other multiple HIF1a-independent pathways, including the mitogen-activated protein kinase pathway and protein stabilisation pathways, which result in the induction of factors such as ATF3 (Ameri et al, 2007;Nemetski and Gardner, 2007) and ATF4 (Ameri et al, 2004;Koditz et al, 2007;Rzymski et al, 2008).…”

Role of ATF4 in regulation of autophagy and resistance to drugs and hypoxia

“…5 However, severe and prolonged hypoxia <0.01% O 2 is considered a functionally different state. 6,7 The endoplasmic reticulum (ER) is highly sensitive to severe hypoxic stress. 8 Protein maturation and disulfide bound formations of proteins destined for secretion and for plasma membranes typically occur in the ER and are oxygendependent 9 ( Fig.…”