Abstract
The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics.
Highlights
Malignant gliomas are highly lethal and devastating diseases that eventually fail to respond to current therapies
To examine the translational components of unfolded protein response (UPR) function, we scrutinized gene expression data of polyribosomebound mRNA of xenograft tumors and parent cell lines, whereupon we demonstrated that the UPR drives profound chemoresitance when induced in glioma cell lines
To assess UPR activation in glial tumors, glioma cell lines and their corresponding xenograft tumor samples were examined for relative mRNA levels for endoplasmic reticulum (ER) chaperones and UPR-coupled transcription factors
Summary
Malignant gliomas are highly lethal and devastating diseases that eventually fail to respond to current therapies. The UPR is divided into two coordinately regulated responses: (1) an initial attenuation of global protein synthesis to slow the influx of newly synthesized proteins into the secretory pathway, and (2) a transcriptional remodeling event that elevates expression of a cohort of stress response genes [14]. In biological contexts, these downstream events may be provoked simultaneously or may be individually activated. The UPR pushes cells to either “work through” the problem—leading to recovery from the stress--or the cells undergo apoptosis if the stress is insurmountable
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