High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor

Abstract

BackgroundEpidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma.MethodsExpression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown.ResultsEREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α.ConclusionEREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition

“…It is important for evaluation of possible signifi cance of these genes in the control of glioma growth through ER stress signaling mediated by IRE1 and hypoxia. It is known that stress signaling pathways are involved in numerous metabolic pathways and inhibition of the activity of IRE1 signaling enzyme in glioma cells had antitumor eff ects (Auf et al 2010(Auf et al , 2013Manie et al 2014;Minchenko et al 2015).…”

“…Th e changes in these genes expression in cells without IRE1 signaling enzyme function possible contribute in the suppression of glioma cell proliferation and tumor growth, because there are data indicating that the estrogen receptor related factors play an important role in the control of cell proliferation and apoptosis (Cho et al 2010;Lapierre et al 2015;Tiwari et al 2015). It is possible that increased expression of FAM162A, PGRMC2, TRIM16 and SLC39A6 genes as well as decreased expression of ESRRA, and NRIP1 genes (Figure 1) may contribute to the suppression of the proliferation and glioma growth from glioma cells with IRE1 knockdown (Auf et al 2010(Auf et al , 2013Aziz et al 2015;Casaburi et al 2015;Minchenko et al 2015;Zhang et al 2015;Matsushima et al 2016). Th erefore, suppressing of NRIP1, which modulates transcriptional activity of the estrogen receptor, as well as knockdown of a site-specifi c transcription regulator ESRRA/NR3B1 inhibits growth of the cancer cells in vitro and in vivo (Aziz et al 2015;Matsushima et al 2016).…”

“…Th e expression level of studied genes in these cells was compared with cells, transfected by vector (control 1), but this subline was also used as control 2 for investigation the eff ect of hypoxia on gene expressions under blockade of both enzymatic activities of IRE1. Th e effi ciency of IRE1 suppression in this glioma cell subline was estimated previously (Auf et al 2010(Auf et al , 2013 by determining the expression level of the XBP1 alternative splice variant, a key transcription factor in the IRE1 signaling, and the level of the phosphorylated isoform IRE1 using cells treated by tunicamycin (0.01 mg/ml during 2 h) for induction of ER stress. Both used in this study sublines of glioma cells are grown with the addition of geneticin (G418) while these cells carrying empty vector pcDNA3.1 or dnIRE1 construct.…”

“…However, involving of endoplasmic reticulum (ER) stress signaling into the regulation of diff erent metabolic processes by estrogens has not been explored yet. Th e ER stress is mediated by three sensor and signaling pathways, but inositol requiring enzyme 1 (IRE1/ERN1) is a central mediator of the unfolded protein response and an important component of tumor growth, because its inhibition leads to a suppression of glioma growth through down-regulation of the angiogenesis and proliferation processes (Drogat et al 2007;Auf et al 2010Auf et al , 2013. Th is stress is recognized as an important determinant of cancer and contributes to the expression profi le of many regulatory genes resulting in proliferation, apoptosis, angiogenesis, and circadian clock (Clarke et al 2014;Manie et al 2014;Pluquet et al 2014;Dejeans et al 2015;Minchenko et al 2015).…”

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

“…Both domains contributed to ERN1 signaling [30]. The IRE1as sociated protein kinase autophosphorylates and di merizes this enzyme in the endoplasmic reticulum membrane, leading to the activation of its endoribo nuclease domain, and has some additional functions [7]. Endoribonuclease activity is responsible for deg radation of a specific subset of mRNA and initiation of the preXBP1 (Xbox binding protein 1) mRNA splicing that stimulates the expression of more than five hundreds of unfolded protein response-specific genes [6,30,31].…”