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Cancer cells are exposed to metabolic stress caused by hypoxia, lactate and acidic pH in the tumor microenvironment. Adaptation to these metabolic stress conditions is required for the cells to survive. A better understanding of the stress responses can lead to new therapeutic strategies and be of clinical importance. We here aimed to determine regulatory networks of transcription factors (TFs) and their target genes in the response to hypoxia (HYP), lactic acidosis (LA) and hypoxia combined with lactic acidosis (HYP-LA) in cervical cancer, and further to identify biological features associated with each response.

The regulatory networks were constructed using Illumina gene expression data from HeLa and SiHa cells exposed to each stress condition, and from tumour biopsies of 280 patients with locally advanced cervical cancer. HYP, LA and HYP-LA metagenes were determined in clinical gene expression data, based on correlations with cell line derived seed genes. iRegulon was used to detect enriched TF motifs in each metagene and construct the regulatory network of each stress response. Biological associations were identified by gene set enrichment analyses of the TF target genes and genes correlating with calculated target gene scores. Stroma fraction in tumor biopsies, hypoxia level images from DCE-MRI (n = 59), small RNA sequencing data for miR-200 (n = 200), a well-known inhibitor of epithelial-mesenchymal transition (EMT), and immunohistochemistry data for HIF1A (n = 261), T-cells (CD8) (n = 260), vascularization (Factor VIII) (n = 247), and EMT (Vimentin) (n = 23) were used to validate gene expression-based findings. TCGA data (n=252) were used for validation in an external cohort.

Significant differences in the regulatory networks were found among the three stress responses. HIF and AP-1 were identified as important TFs for the HYP response. Upregulation of TCF12 and downregulation of SMAD4 was among the most important TFs for LA and HYP-LA, respectively. The HYP response was associated with high expression of glycolysis genes and downregulation of anti-tumor immunity. For the LA response, positive associations with EMT, stroma fraction and vascularization were identified. HYP-LA was associated with severe hypoxia by imaging, upregulation of genes involved in cytoskeleton organization and autophagy, and downregulation of oxidative phosphorylation genes. Gene sets of immunity in the HYP response and EMT in the LA response were associated with aggressive disease.

TFs, target genes and biological features differ considerably among the three stress responses in cervical cancer. Immune and stroma cells seem to be important in the aggressive tumor phenotype associated with the responses to hypoxia and lactic acidosis, respectively.