Inhibition of kinase and endoribonuclease activity of ERN1/IRE1α affects expression of proliferationrelated genes in U87 glioma cells

Abstract

Inhibition of ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme-1α), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F1), and HOXC6 (homeobox C6) in U87 glioma cells overexpressing dominant-negative ERN1/IRE1α defective in endoribonuclease (dnr-ERN1) as well as defective in both kinase and endonuclease (dn-ERN1) activity of ERN1/IRE1α. We have demonstrated that the expression of all studied genes is decreased at the mRNA level in cells with modified ERN1/IRE1α; TBX3, however, is increased in these cells as compared to control glioma cells. Changes in protein levels of E2F8, HOXC6, ATF3, and TBX3 corresponded to changes in mRNAs levels. We also found that two mutated ERN1/IRE1α have differential effects on the expression of studied transcripts. The presence of kinase and endonuclease deficient ERN1/IRE1α in glioma cells had a less profound effect on the expression of E2F8, HOXC6, and TBX3 genes than the blockade of the endoribonuclease activity of ERN1/IRE1α alone. Kinase and endonuclease deficient ERN1/IRE1α suppresses ATF3 and FOXF1 gene expressions, while inhibition of only endoribonuclease of ERN1/IRE1α leads to the up-regulation of these gene transcripts. The present study demonstrates that fine-tuning of the expression of proliferation related genes is regulated by ERN1/IRE1α an effector of endoplasmic reticulum stress. Inhibition of ERN1/IRE1α, especially its endoribonuclease activity, correlates with deregulation of proliferation related genes and thus slower tumor growth.

Inhibition of IRE1 modifies effect of glucose deprivation on the expression of TNF?-related genes in U87 glioma cells

“…3, c). These results correlate well with our previous data that the inhibition of IRE1 up-regulates TP53 expression [48] and glucose deprivation can contribute to this pro-apoptotic effect of IRE1 inhibition. Thus, an increased expression of lItAF gene can induce TNFα expression and TNFα-mediated apoptosis.…”

“…1, A). Moreover, the changes observed in the above studied gene, which has relation to TNF-directed apoptosis, correlate well with slower cell proliferation in cells harboring dn-IRE1 [10,18,48], attesting to the fact that endoplasmic reticulum stress is a necessary component of malignant tumor growth and cell survival [2,3,6]. There is data that TNFRSF21/DR6 induced apoptosis through a new pathway that is different from the type I and type II pathways through interacting with Bax protein [19].…”

Inhibition of IRE1 modifies hypoxic regulation of G6PD, GPI, TKT, TALDO1, PGLS and RPIA genes expression in U87 glioma cells

“…It is well known that GPI/AMF has proproliferative properties because it contributes to energy pathways and as cytokine (not enzymatic activity) and its si lencing resulted in mesenchymaltoepithelial transi tion of human cancer cells with reduced malignancy [2426]. Thus, our results are mostly consistent with numerous data [9,10,27,40] that hypoxia associated with malignant progression through the endoplasmic reticulum unfolded protein response, but mecha nisms through which malignant cells cope with po tentially lethal metabolic stress induced by hypoxia remains poorly understood. At the same time, the expression of two other genes (G6PD and tkt) in control glioma cells is re sistant to hypoxic treatment, but the expression level of TALDO1 and rPIA genes is decreased.…”

“…In this study we used sublines of U87 glioma cells, which were described previously [5,9]. One subline was obtained by selection of stable transfec ted clones with overexpression of vector pcDNA3.1, which was used for creation of dnIRE1 (dominant/ negative IRE1).…”

“…Total RNA was extracted from glioma cells using Trizol reagent according to man ufacturer protocol (Invitrogen, USA) as described previously [7,9]. The RNA pellets were washed with 75% ethanol and dissolved in nucleasefree wa ter.…”