The unfolded protein response increases production of pro-angiogenic factors by tumor cell lines

The rapid growth and proliferation of tumor cells will be limited at a stage when they encounter inadequate levels of oxygen and nutrient supply within the poorly vascularized tumor mass. These severe conditions negatively affect the proper folding of nascent proteins in the endoplasmic reticulum (ER) and lead to accumulation of unfolded protein within ER which is referred to as ER stress. Consequently, it will trigger the unfolded protein response (UPR) signal pathway through ER membrane stress sensor proteins including activating transcription factor 6 (ATF6), inositol-requiring 1 (IRE1) and PKR-like ER localized kinase (PERK). The UPR is largely a cytoprotective response and is thought to contribute to tumor survival in the face of inadequate nutrients and oxygen. Microarray analyses were conducted on Daoy, a human medulloblastoma line that was treated with thapsigargin, which activates the UPR by depleting Ca2+ from the ER. In addition to the expected UPR targets, we found that ER stress inducing agents led to the transcriptional induction of several pro-angiogenic factors including vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), interleukin 8 (IL-8) and angiogenin. Using quantitative real-time PCR, we confirmed that a number of UPR inducing conditions (i.e., thapsigargin, tunicamycin, and no glucose) up-regulated VEGF, IL-8 and Angiogenin transcripts and extended these finding to a rat glioma line, a mouse fibroblast line and two human neuroblastoma lines. Our western blot and ELISA assay demonstrated the protein and secretion levels of VEGF were also elevated in C6 cells under ER stress condition.;To understand the mechanism by which ER stress triggers the up-regulation of pro-angiogenic factors, we tested the transcription rate and mRNA half-life of VEGF under ER stress condition. Both are dramatically increased by thapsigargin and glucose deprivation in C6 rat glioma cells. By chromatin IP experiments, we found that XBP1 bound to the promoter region of VEGF gene in response to ER stress in C6 cells which suggested that XBP1 may transactivate VEGF gene during UPR activation. After testing several stress inducible kinases which have been shown to contribute to stabilize VEGF mRNA in different cell lines in response to various stress conditions, we found that activation of both AMP-activating protein kinase (AMPK) and p38 mitogen activating protein kinase (p38 MAPK) elevate VEGF mRNA level by increasing its stability during ER stress. We also found that activation of JNK increase VEGF mRNA by increasing its transcription in response to the UPR. These results suggest that ER stress may increase the production of pro-angiogenic factors at multiple levels including increasing transcription of VEGF and stabilization of its mRNA, thus contributing to tumor angiogenesis.