Inhibition of ERN1 (endoplasmic reticulum to nuclei 1), the major signalling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of tumor protein 53 (TP53)- related genes such as TOPORS (topoisomerase I binding, arginine/serine-rich, E3 ubiquitin protein ligase), TP53BP1 (TP53 binding protein 1), TP53BP2, SESN1 (sestrin 1), NME6 (non-metastatic cells 6), and ZMAT3 (zinc finger, Matrin-type 3) in glioma cells expressing dominantnegative ERN1 under baseline and hypoxic conditions. We demonstrated that inhibition of ERN1 function in U87 glioma cells resulted in increased expression of RYBP, TP53BP2, and SESN1 genes, but decreased expression of TP53BP1, TOPORS, NME6, and ZMAT3 genes. Moreover, inhibition of ERN1 affected hypoxia-mediated changes in expression of TP53-related genes and their magnitude. Indeed, hypoxia has no effect on expression of TP53BP1 and SESN1 in control cells, while resulted in increased expression of these genes in cells with inhibited ERN1 function. Magnitude of hypoxia-mediated changes in expression levels of RYBP and TP53BP2 was gene specific and more robust in the case of TP53BP2. Hypoxiamediated decrease in expression levels of TOPORS was more prominent if ERN1 was inhibited. Present study demonstrates that fine-tuning of the expression of TP53- associated genes depends upon endoplasmic reticulum stress signaling under normal and hypoxic conditions. Inhibition of ERN1 branch of endoplasmic reticulum stress response correlates with deregulation of p53 signaling and slower tumor growth.

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Endoplasmic reticulum stress, as well as hypoxia and ischemia, are important factors for tumor neovascularization and growth. Cancer cells preferentially utilize glycolysis in order to satisfy their increased energetic and biosynthetic requirements. High glucose metabolism of cancer cells is caused by a combination of hypoxia-responsive transcription factors, activation of oncogenic proteins and the loss of tumor suppressor function and is realized in part by activating a family of regulatory bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) and hexokinase 2. We have studied the effect of hypoxia and ischemia on the expression of PFKFB and hexokinase genes in glioma cell line U87 under knockdown of endoplasmic reticulum-nuclei-1 (ERN1) sensing and signaling enzyme. It was shown that loss of the signaling enzyme ERN1 function leads to an increase in the expression levels of HK1, HK2, PFKFB3 and PFKFB4 mRNA. Moreover, the expression levels of all studied genes increase under hypoxia in control and ERN1-deficient glioma cells; however knockdown of ERN1 suppresses the effect of hypoxia. Besides, HK2 and PFKFB4 are more sensitive to hypoxia than HK1 and PFKFB3. Glucose or glutamine deprivation conditions have different effects on the expression levels of these genes and its effect depends mainly on ERN1 function. Expression levels of alternative splice variants of PFKFB3 and PFKFB4 mRNA change at used experimental conditions in a fashion similar to the basic PFKFB variants. Thus, the expression of hexokinase and PFKFB genes is mainly dependent on ERN1 signaling enzyme function in normal, hypoxic and ischemic conditions.

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