ATF4 and ATF6 orchestrate transcriptional responses to glucose deprivation in NSCLC

Tumours grow faster than the local vasculature, resulting in a shortage of oxygen and nutrients. Adaptation involves signal transduction and metabolic, translational and transcriptional changes. We analysed transcriptional responses to glucose deprivation using RNA sequencing of A549 cells to identify regulation of individual genes and pathways in Reactome through gene-set-enrichment analysis. The most relevant transcription factors inferred by bioinformatic analysis, ATF4 and ATF6, were silenced using siRNA, and gene expression and cell death was studied in several lines of non-small cell lung carcinoma (NSCLC). Amino acids and metabolites of glycolysis and the TCA were measured upon glucose deprivation and in ATF4-silenced cells. Multiple pathways related to DNA replication and cell cycle were downregulated upon glucose deprivation. The highest differentially upregulated transcriptional pathway was the Unfolded Protein Response (UPR), which has been shown to promote adaptation to proteotoxic and endoplasmic reticulum stress. 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. ATF6 and ATF4 were the transcription factors with a higher score upon network analysis. Multiple metabolic genes and pathways were regulated by ATF4 or ATF6 siRNA, including pathways of amino acid synthesis and transport, glycosylation and lipid synthesis. ATF4 silencing also regulated the transcriptional induction of mitochondrial OXPHOS-associated pathways. Metabolite analysis revealed that ATF4 regulates amino acid and citrate levels during glucose deprivation. Functionally, rather than survival, ATF4 contributed to cell death, with limited contribution of its target DR5/TRAILR2. Silencing CHOP, DR4 or NOXA did not affect cell death in glucose-starved A549 cells. Similarly, silencing or inhibiting other UPR factors like ATF6 and IRE1/XBP1s individually or in combination did not impact survival upon glucose deprivation. Of note, the absence of ATF4 or sestrin 2 prevented mTORC1 inactivation upon glucose deprivation. In contrast, ATF6 reduction enhanced the activation of XBP1 and ATF4 but it did not regulate mTORC1 inactivation. These findings indicate that the UPR is activated upon glucose deprivation and that some of the UPR transcription factors control transcriptional and metabolic responses to glucose shortage.