ATF4 and ATF6 produce diverse transcriptional signatures affecting metabolic genes and cell death under glucose deprivation

Tumours grow faster than the local vasculature, resulting in a shortage of oxygen and nutrients. Among nutrients reduced in the tumor microenvironment, glucose is essential for tumour growth and survival. To explore how lung cancer cells cope with a low-glucose environment, we analysed transcriptional changes in glucose-starved A549 cells using RNA-sequencing. This showed downregulation of multiple pathways related to DNA replication and cell cycle. Many pathways were upregulated, and among them, the most upregulated transcriptional response to glucose deprivation was the Unfolded Protein Response (UPR), which has been shown to promote adaptation to proteotoxic and endoplasmic reticulum stress. The UPR involves three signalling branches, the PERK/ATF4, IRE1/XBP1, and ATF6 pathways. Glucose shortage robustly induced the three branches of the UPR in several non-small cell lung carcinoma cell lines, as indicated by ATF4 accumulation, XBP1 mRNA splicing and ATF6 cleavage. Transcriptional network analysis indicated that the transcriptional response to glucose deprivation was primarily driven by ATF6 and ATF4. Their silencing revealed that they cooperate to regulate multiple metabolic genes and pathways related to lipid synthesis and to amino acid synthesis and transport. ATF4 additionally regulated the transcriptional induction of mitochondrial OXPHOS-associated pathways. Functionally, ATF4 contributed to cell death, while ATF6 and IRE1/XBP1s did not impact survival. Interestingly, the ATF4 targets CHOP, Noxa and DR4 (TRAIL-R1) did not mediate cell death, which was only partially dependent on TRAIL-R2 (DR5) and only partially prevented by caspase inhibitors. Of note, both ATF4 and ATF6 regulated CHOP, and the absence of ATF6 enhanced the activation of XBP1 and ATF4 under glucose deprivation. These findings indicate that glucose deprivation initiates a complex interplay between the different branches of the UPR, which shape the balance between metabolic homeostasis and cell death.