Some tumours contain regions of very severe hypoxia called anoxia, which constitutes a functionally different state to hypoxia. In response to anoxia, mammalian cells induce coordinated cytoprotective programs that are critical for tumour survival-the unfolded protein response (UPR) and integrated stress response (ISR). ATF4 is integral for anoxia and regulates the ISR, a signalling pathway initiated by phosphorylation of eIF2α by PERK and protects cells against metabolic consequences of ER stress. We investigated the role of ATF4 in this process..

We compared polysome-bound and total RNA isolated from aerobic-normoxic (0h) and anoxic (24h, 48h) ATF4 siRNA knock-down MCF7 cells using the Illumina 48k human BeadArray Chips. This analysis revealed that genes involved in translational control, amino acid metabolism, cell adhesion, proliferation, downstream transcription factors and chaperones were specifically induced under anoxia in an ATF4 dependent manner. We also determined a novel group of anoxia-induced genes regulated by changes in the rate of translation of mRNA under anoxia. Microarray analysis also showed that a substantial number of HIF1α target genes remained upregulated under prolonged anoxia (48h) although after initial induction, HIF1α was down regulated and undetectable at 24-48 hours. In addition, several genes were found to be upregulated HIF1α and downregulated by ATF4, including HIG2, RBPSUH, PDK3 and EPHA2. Therefore we speculated that under chronic anoxia ATF4 replaced and antagonised HIF1α on the sites of transcription. We validated anoxia and ATF4 dependent genes and determined the time-course induction by qPCR and immunoblotting analysis. One of the most profound cytoprotective measures under anoxia is the repression of Cyclin D1 and arrest at the G1/S phase of the cell cycle. We found and proved that repression of Cyclin D1 under anoxia is mediated by ATF4. In addition, ATF4 stimulated the anoxic expression of CCNDBP1, a negative regulator of Cyclin D1 dependent expression. Knock down of ATF4 resulted in cell death after 24 hours of exposure to anoxia, whereas proliferation and survival of normoxic cells remained unchanged. Therefore it is likely that cell cycle arrest by downregulation of cyclin D1 was a major pathway of survival under anoxia

Other groups of ATF targets included genes involved in the translational control under anoxia. We also validated anoxic induction of PERK (eIF2AK3), negative regulators of cap-dependent translation: EIF4EBP1, DCP2, PAIP; several tRNA synthetases and tRNA transporter XPOT were all mediated by ATF4.

These results indicated that the mechanism of anoxia-induced translational attenuation and control could be linked to ATF4 and that adaptation of cells to anoxic stress required activation of ATF4.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA