ExprEssion of ubiquitin spEcific pEptidasE and ATG 7 gEnEs in u 87 glioma cElls upon glutaminE dEprivation

We have studied the effect of glutamine deprivation on the expression of genes encoding for ubiquitin specific peptidases (usP) and ubiquitin activating enzyme E1-like protein/autophagy related 7 (Gsa7/atG7) in u87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (iRE1). it was shown that exposure of control glioma cells (transfected by empty vector) upon glutamine deprivation led to suppression of usP1 and atG7 mRna expression and up-regulated usP25 mRna. at the same time, glutamine deprivation did not significantly change usP4, usP10, usP14, and usP22 gene expressions in these cells. inhibition of ІRE1 signaling enzyme function in u87 glioma cells increased effect of glutamine deprivation on the expression of usP1 gene and introduced sensitivity of usP4 and usP14 genes to this condition. therefore, glutamine deprivation affected the expression level of most studied genes in gene specific manner in relation to the functional activity of iRE1 enzyme, a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth.

We have studied the effect of glutamine deprivation on the expression of genes encoding for ubiquitin specific peptidases (usP) and ubiquitin activating enzyme E1-like protein/autophagy related 7 (Gsa7/atG7) in u87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (iRE1).it was shown that exposure of control glioma cells (transfected by empty vector) upon glutamine deprivation led to suppression of usP1 and atG7 mRna expression and up-regulated usP25 mRna.at the same time, glutamine deprivation did not significantly change usP4, usP10, usP14, and usP22 gene expressions in these cells.inhibition of ІRE1 signaling enzyme function in u87 glioma cells increased effect of glutamine deprivation on the expression of usP1 gene and introduced sensitivity of usP4 and usP14 genes to this condition.therefore, glutamine deprivation affected the expression level of most studied genes in gene specific manner in relation to the functional activity of iRE1 enzyme, a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth.K e y w o r d s: mRna expression, usPs, atG7, glutamine deprivation, iRE1 inhibition, u87 glioma cells.M alignant gliomas are highly aggressive tumors with very poor prognosis and to date there is no efficient treatment availab le.The moderate efficacy of conventional clinical approaches therefore underlines the need for new therapeutic strategies.Glutamine is important to glio ma development and a more agressive behaviour [1][2][3][4].However, mechanisms whereby cancer cells regulate glutamine catabolism remain largely unknown [4][5][6].A better knowledge of tumor respon ses to glutamine deprivation condition is required to elaborate new therapeutical strategies of cell sensibilization, based on the blockade of survival mechanisms.
Ubiquitin is a highly conserved protein involved in regulation of intracellular protein breakdown, cell cycle regulation, chromatin remodeling, and stress response.It is released from degraded proteins by disassembly of the polyubiquitin chains, which is mediated by ubiquitin-specific proteases, members of the ubiquitin-specific processing fami-ly of proteases for deubiquitination of proteins [7][8][9].E3 ubiquitin ligases and deubiquitylases play an important role in cancer [7,10,11].Our previous results demonstrated possible interaction/cross-talk between unfolding protein response signaling and ubiquitin system during adjustment to episodes of hypoxia during tumor development [12].Ubiquitin specific peptidases (USPs) and ubiquitin activating enzyme E1-like protein/autophagy related 7 (GSA7/ ATG7) are involved in cancer cells survival and progression [13][14][15].USP1 and USP7 are responsible for deubiquitination of mono-ubiquitinated PCNA (proliferating cell nuclear antigen), which activates error-prone DNA polymerases and controls an oxidative-stress-induced mutagenesis in human cells [13].Decreased levels of USP1 in cancer cells have been implicated in lung and glioblastoma tumors growth and progression [14,16].There is data that serine phosphorylation is critical for the activity of USP1 and its interaction with WD40-repeat protein UAF1; while two nuclear localization signals in USP1 media te nuclear import of the USP1/UAF1 complex [17].
Ubiquitin specific peptidase 4 function is important during tumorigenesis because this deubiquitinating enzyme has a key role in the regulation of TP53 and TGFβ signaling and is also a positive regulator of the WNT/β-catenin signaling [18][19][20].Deubiquitinating enzyme UPS10 suppresses the proliferation and growth of cancer cells through stabilization of p53 protein [21].Additional anti-tumorigenic effect of USP10 achieved by antagonizing c-MYC activity through stabilization of a tumor suppressor SIRT6 [22].In agreement, microRNA-191 mediated lower protein level of USP10 has been demonstrated to promote pancreatic cancer progression [21].It was shown that USP14 is a tumor promoting peptidase, its phosphorylation and activation by Akt not only regulates the ubiquitin-proteasome system, but also promotes tumor progression through regulation of cellular proliferation and apoptosis of cancer cells [23].Inhibition of USP14 could be used as potential anti-cancer therapeutic strategy.USP22 protease has been demonstrated to participate in regulation of the cell cycle progression in many cancer cell types [24,25].This enzyme removes ubiquitin from histones, thus regulating gene transcription [26].It is interes ting to note that deubiquitinating enzyme USP25 is involved in endoplasmic reticulum (ER)associa ted degradation (ERAD) of misfolded/anomalous proteins [27].USP25 counteracts ubiqui tination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome [27].USP25 is a novel TRiC interac ting protein that is catalyzed deubiquitination of the TRiC protein and stabilized this chaperonin, thereby reducing accumulation of misfolded protein aggregates [28].
The ubiquitin activating enzyme E1-like protein (GSA7), which is also known as autophagy related 7 (ATG7), is an essential component of autophagic machinery and a multifunctional protein, which mediates inhibition of cell proliferation and activation of apoptosis through induction of cellular senescence [29][30][31][32].Thus, autophagy inhibition by Baf A1 or knockdown of ATG7 or ATG12 induced cytotoxicity in multiple human bladder cell lines.Induction of apoptosis was found in cells with autophagy inhibition [32].Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in pro-tein synthetic capacity [33].Moreover, knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel endoplasmic reticulum stress-induced death pathway [34].The endoplasmic reticulum stress is responsible for enhanced cancer cell proliferation and IRE1 knockdown by a dominant-negative construct of IRE1 (dnIRE) resulted in a significant anti-proliferative effect on glioma growth [35,36].
The rapid growth of solid tumors generates micro-environmental changes in association to hypoxia, nutrient deprivation and acidosis, which promote neovascularisation, cell survival and proliferation [37][38][39][40].Glucose and glutamine are substrates for glycolysis and glutaminolysis, which are important for tumor progression through regulation of the cell cycle at distinct stages [1][2][3][4][5].The activation of glycolysis and glutaminolysis in cancer cells is tightly regulated by the action of two ubiquitin ligases, which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase-3 (PFKFB3) and glutaminase 1 (GLS1), the first enzyme in glutaminolysis [1,4,6].The activation of endoplasmic reticulum stress is indispensable for tumor growth as it facilitates adaptation to stressful environmental conditions [40].IRE1 is the most evolutionary conserved sensor that responds to protein misfolding with a highly tuned program aimed to either resolve the stress or direct the cell towards apoptosis in case stress becomes too severe, which makes it a key regulator of cell life and death processes [35,40].Recently, we have shown that glutamine deprivation affects the expression of proliferation related genes in U87 glioma cells and that IRE1 knockdown modifies glutamine deprivation effects on these genes expression possibly contributing to suppression of glioma cells proliferation [41].Previous ly, we have shown that most USPs are regulated by IRE-1α signaling and hypoxia as well as glucose deprivation [42][43][44], but the precise mechanism of the exhibited by USP7 anti-proliferative effect is not clear yet.
The aim of this study was investigation of the effect of glutamine deprivation condition on the expression of a subset of genes encoding ubiquitin specific peptidases and of ubiquitin activating enzyme E1-like protein/autophagy related 7 in glioma cells in relation to inhibition of signaling enzyme IRE1 with hopes of elucidating its mechanistic part in the development and progression of certain cancers and the contribution to unfolding protein response.

materials and methods
cell lines and culture conditions.Two sublines of U87 glioma cells were used in this study.Cells are growing in high glucose (4.5 g/l) Dulbecco's modified Eagle's minimum essential medium (DMEM; Gibco, Invitrogen, USA) supplemented with glutamine (2 mM), 10% fetal bovine serum (Equitech-Bio, Inc., USA), streptomycin (0.1 mg/ml; Gibco) and penicillin (100 units/ml; Gibco) at 37 °C in a 5% CO 2 incubator.One subline was obtained by selection of stable transfected clones with overexpression of empty vector (pcDNA3.1),which was used for creation of dnIRE1 (dominant-negative constructs of IRE1, bifunctional sensing and signaling enzyme of endoplasmic reticulum stress).This untreated subline of glioma cells (control glioma cells) was used as control in the study of effects of glutamine deprivation on the expression level of USP1, USP4, USP10, USP22, USP25, and Gsa7 genes.Second subline was obtained by selection of stable transfected clones with overexpression of dnIRE1 and has suppressed both protein kinase and endoribonuclease activities of this enzyme.The expression level of studied genes in these cells was compared with cells, transfected by vector.The subline, which overexpress dnIRE1, was also used as control for investigation the effect of glutamine deprivation condition on the expression level of studied in cells with inhibited signaling enzyme IRE1 function.U87 glial cells clone with dnIRE1 was received by selection at 0.8 mg/ml geneticin (G418) and grown in the presence of this antibiotic at lower concentration (0.4 mg/ ml).Glutamine deprivation conditions were created by changing the complete DMEM medium into culture plates on medium without glutamine and plates were exposed to this condition for 16 h.
The suppression level of IRE1 both enzymatic activity in glioma cells that overexpress a dnIRE1 was estimated previously [36] by determining the expression level of XBP1 alternative splice variant (XBP1s), a key transcription factor in IRE1 signaling , using cells treated by tunicamycin (0.01 mg/ml during 2 h).Moreover, the proliferation rate of glioma cells with mutated IRE1 is decreased more than in 2 fold [36].Thus, the blockade of signaling enzyme IRE1 activity has significant effect on proliferation rate of glioma cells.
Rna isolation.Total RNA was extracted from glioma cells as previously described [43].The RNA pellets were washed with 75% ethanol and dissolved in nuclease-free water.For additional purification RNA samples were re-precipitated with 95% ethanol and re-dissolved again in nuclease-free water.RNA concentration and spectral characteristics were measured using NanoDrop Spectrophotometer.
The primers were received from Sigma-Aldrich (St. Louis, MO, USA).The quality of amplification products was analyzed by melting curves and by electrophoresis using 2% agarose gel.An analysis of quantitative PCR was performed using special computer program "Differential Expression Calculator".The values of USP1, USP4, USP10, USP22, USP25, and GSA7 mRNA expressions were normalized to the expression of beta-actin mRNA and represented as percent of control 1 (100%).
Statistical analysis.All values are expressed as mean ± SEM from triplicate measurements performed in 4 independent experiments.Statistical analysis was performed according to Student's t-test using Excel program as described previously [45].

results and discussion
To determine if glutamine deprivation regulates the genes of interest through the IRE1 branch of endoplasmic reticulum stress response, we investigated the effect of glutamine deprivation condition on the expression of genes encoding USP1, USP4, USP10, USP22, USP25, and GSA7 in two sublines of U87 glioma cells in relation to inhibition of IRE1 signa ling enzyme, which is a major component of the unfolded protein response.As shown in Fig. 1, the exposure of control glioma cells (transfected by empty vector) upon glutamine deprivation condition leads to small but statistically significant suppression of USP1 and GSA7 mRNA expression (-13 and -19%, correspondingly) as compared to control glio ma cells.At the same time, the expression level of four other genes of USPs (and USP22) does not change significantly in control glioma cells treated by glutamine deprivation, but USP25 gene expression is up-regulated (+22%) under this experimental condition as compared to control glioma cells (Fig. 1).
As shown in Fig. 2, glutamine deprivation does not significantly change the level of USP10 and USP22 genes expression in glioma cells without IRE1 signaling enzyme function in comparison with corresponding control cells.At the same time, the expression level of USP25 gene is increased (+17%) in dnIRE1 glioma cells treated by glutamine deprivation (Fig. 2).Therefore, inhibition of IRE1 signaling enzyme function in U87 glioma cells by dnIRE1 does not change significantly the sensitivity of USP10, USP22, and atG7 gene expression to glutamine deprivation and introduces sensitivity of USP4 and USP14 gene expressions to this experimental condition, as shown in Fig. 3 and 4.
Thus, inhibition of IRE1 modifies the sensitivity of USP1, USP4 and USP14 gene expressions to glutamine deprivation in U87 glioma cells.As shown in Fig. 3, the suppression of USP1 gene expression by  glutamine deprivation is more significant in glioma cells transfected by dnIRE1 as compared to control glioma cells.Moreover, the expression of USP4 and USP14 genes, which is resistant to glutamine deprivation in control glioma cells, is decreased after inhibition of IRE1 signaling enzyme function (Fig. 3  and 4).At the same time, IRE1 knockdown does not change significantly sensitivity of USP10, USP25, and Gsa7 gene expressions to glutamine deprivation condition in these glioma cells (Fig. 3 and 4).

Fig. 3. Comparative effect of glutamine deprivation on the expression level of usP1, usP4, and usP10 mRna in two types of glioma cells: control cells transfected by vector (vector) and cells with a deficiency of the signaling enzyme iRE1 (dniRE1) measured by qPCR. values of these mRna expressions were normalized to β-actin mRna expression and represented as percent of corresponding control (control for both cell types is accepted as 100%); mean ± sEM; n = 4
In this study we have shown that the expression of USP4, USP10, USP14, and USP22 genes are resistant to glutamine deprivation condition in control (transfected by empty vector) glioma cells because the exposure of cells to glutamine deprivation does not significantly change the level of their expression.In control glioma cells glutamine deprivation affects the expression of USP1 and USP25 genes only.It is possible that the resistance of most studied USPs to glutamine deprivation can be associated with important functions of these enzymes in metabolic processes, cell proliferation and surviving [7-9, 13, 14].Inhibition of ІRE1 signaling enzyme function in U87 glioma cells increased effect of glutamine deprivation on the expression of USP1 gene and introduced sensitivity of USP4 and USP14 genes to this condition.A decreased level of USP1, USP4, and USP14 mRNA expression upon glutamine deprivation agrees well with functional role of these enzymes and suppression of glioma cell proliferation, because there is data that USP1 targeting impedes GBM growth and that USP4 and USP14 regulate cellular proliferation and apoptosis [16,18,23].

Fig. 4 .
Fig. 4. Comparative effect of glutamine deprivation (16 h) on the expression level of usP14, usP22, usP25, and atG7/Gsa7 mRna in two types of glioma cells: control cells transfected by vector (vector) and cells with a deficiency of the signaling enzyme iRE1 (dniRE1) measured by qPCR.values of these mRna expressions were normalized to β-actin mRna expression and represented as percent of corresponding control (control for both cell types is accepted as 100%); mean ± sEM; n = 4