ATF4 and ATF6 Orchestrate Transcriptional Responses to Glucose Deprivation in NSCLC

Background Tumours grow faster than the local vasculature, resulting in a shortage of oxygen and nutrients. Adaptation to nutrient shortages involves signal transduction and metabolic, translational and transcriptional changes. Methods We analysed transcriptional responses to glucose deprivation using RNA sequencing of A549 cells to identify regulation of individual genes and metabolic pathways in Reactome through gene-set-enrichment analysis. The most relevant transcription factors inferred by bioinformatic analysis were silenced using siRNA and results studied in several lines of non-small cell lung carcinoma (NSCLC). Results 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. Network analysis unveiled ATF6 and ATF4 as the most relevant transcription factors. siRNA revealed that these factors 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, rather than survival, ATF4 contributed to cell death, with limited contribution of its targets Noxa, DR4 and DR5. Other UPR factors like ATF6, CHOP -upregulated by both ATF4 and ATF6- and IRE1/XBP1s did not impact survival. Of note, the absence of ATF4 prevented mTORC1 inactivation upon glucose deprivation. In contrast, ATF6 reduction enhanced the activation of XBP1 and ATF4, but it did not regulate mTORC1 inactivation. Conclusions These findings indicate that the UPR controls transcriptional responses to glucose shortage, and that cooperation and compensation between the main transcription factors further influence nutrient sensing, metabolism and cell death.