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The ERN1 (endoplasmic reticulum to nucleus signaling 1) pathway plays an important role in the regulation of gene expression in glioblastoma, but molecular mechanism has not yet been fully elucidated. The aim of this study was to evaluate the relative relevance of ERN1 activity as a kinase in comparison to its endoribonuclease activity in the regulation of homeobox gene expression. Two sublines of U87MG glioblastoma cells with different ways of ERN1 inhibition were used: dnERN1 (overexpressed transgene without protein kinase and endoribonuclease) and dnrERN1 (overexpressed transgene with mutation in endoribonuclease). ERN1 suppression was also done using siRNA for ERN1. Silencing of XBP1 mRNA by specific siRNA was used for suppression of ERN1 endoribonuclease function mediated by XBP1s. The expression levels of homeobox genes and microRNAs were evaluated by qPCR. The expression of TGIF1 and ZEB2 genes was downregulated in both types of glioblastoma cells with inhibition of ERN1 showing the ERN1 endoribonuclease-dependent mechanism of their regulation. However, the expression of PBX3 and PRPRX1 genes did not change significantly in dnrERN1 glioblastoma cells but was upregulated in dnERN1 cells indicating the dependence of these gene expressions on the ERN1 protein kinase. At the same time, the changes in PAX6 and PBXIP1 gene expressions introduced in glioblastoma cells by dnrERN1 and dnERN1 were different in direction and magnitude indicating the interaction of ERN1 protein kinase and endoribonuclease activities in regulation of these gene expressions. The impact of ERN1 and XBP1 silencing on the expression of studied homeobox genes is similar to that observed in dnERN1 and dnrERN1 glioblastoma cells, correspondingly. The expression of TGIF1 and other homeobox genes is dependent on the ern1 signaling pathways by diverse mechanisms because inhibition of ERN1 endoribonuclease and both ERN1 enzymatic activities had dissimilar impacts on the expression of most studied genes showing that ERN1 protein kinase plays an important role in controlling homeobox gene expression associated with glioblastoma cell invasion. [Display omitted] • Inhibition of ERN1 suppresses TGIF1 and ZEB2 expressions via ERN1 endoribonuclease. • ERN1 knockdown affects PBX3 and PRRX1 gene expressions through ERN1 protein kinase. • The changes in PAX6 gene expression are mediated by ERN1 endoribonuclease and kinase. • ERN1 protein kinase pathway is an important regulator of homeobox genes expressions. [ABSTRACT FROM AUTHOR]

It was shown previously that inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) pathway, a central mediator of the unfolded protein response, leads to suppression of tumor growth through down-regulation of key pro-proliferative and up-regulation of tumor suppressor factors and modifies the sensitivity of these genes to glucose and glutamine deprivation. However, the executive mechanisms of ERN1 mediated control of glioma cell proliferation are not yet known. The goal of this study was to estimate the effect of glucose and glutamine deprivations on expression of cancer related genes in glioma U87 cells at ERN1 signaling inhibition for evaluation of their possible significance in ERN1 mediated control of glioma cell proliferation. We have studied the effect of glucose and glutamine deprivations on the expression level of cancer related genes encoding TMED10 (transmembrane p24 trafficking protein 10), MYL9 (myosin, light chain 9, regulatory), SPOCK1 (sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1), CUL4A (cullin 4A), and CUL4B in U87 glioma control cells and cells with ERN1 knockdown. It was shown that at glucose deprivation the expression level of MYL9, SPOCK1 and CUL4B genes was significantly up-regulated in control glioma cells. ERN1 knockdown modified the sensitivity to glucose deprivation of all studied genes except TMED10 gene. At glutamine deprivation the expression of MYL9, CUL4A and CUL4B genes was shown to be up-regulated in control glioma cells. The sensitivity of MYL9, TMED10 and CUL4B gene expression to glutamine deprivation in glioma cells with ERN1 knockdown was significantly modified, while CUL4A and SPOCK1 gene expression did not respond to ERN1 inhibition. The present study demonstrates that glucose and glutamine deprivation affected the expression of the most studied genes in a specific manner and that inhibition of ERN1 signaling preferentially modified their expression at glucose and glutamine deprivation.

Objective. The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation.

The expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with ERN1 (endoplasmic reticulum to nucleus signaling 1) loss of function as well as upon glutamine deprivation was studied. It was shown that glutamine deprivation down-regulated the expression level of ATF6 (activating transcription factor 6), EIF2AK3/PERK (eukaryotic translation initiation factor 2 alpha kinase 3), GLO1 (glyoxalase I), BIRC5 (baculoviral IAP repeat-containing 5), and RAB5C (RAB5C, a member of RAS oncogene family) mRNAs in control glioma cells. At the same time, the expression level of HSPB8 (heat shock 22kDa protein 8) and HSPA5/GRP78 (heat shock protein family A (Hsp70) member 5) mRNAs was resistant to glutamine withdrawal in these glioma cells. It was also shown that inhibition of ERN1, which controled cell proliferation and tumor growth, modified the effect of glutamine deprivation on the expression levels of most studied genes in U87 glioma cells: up-regulated the expression of ATF6 and HSPA5 genes and enhanced sensitivity of EIF2AK3 and BIRC5 genes to glutamine withdrawal. Furthermore, the expression of all studied genes, except EIF2AK3, was down-regulated in ERN1 knockdown glioma cells in the presence of glutamine. It was demonstrated that glutamine deprivation affected the expression of most studied genes in ERN1 depen­dent manner and that these changes possibly contributed to the suppression of glioma growth from cells without ERN1 signaling enzyme function.