The unfolded protein response in glioblastoma stem cells: towards new targets for therapy
Peñaranda Fajardo, Natalia
DOI:
10.33612/diss.118411504
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Publication date: 2020
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Peñaranda Fajardo, N. (2020). The unfolded protein response in glioblastoma stem cells: towards new targets for therapy. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.118411504
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in Glioblastoma neurospheres
Natalia M. Peñaranda Fajardo1
Cristian Ruiz-Moreno2
Coby Meijer1
Frank A. E. Kruyt1
1Departament of Medical Oncology, University of Groningen,
Univerisity Medical Center Groningen; Groningen, The Netherlands
2Neuroscience Research Group, Medical Research Institute,
Faculty of Medicine, University of Antioquia; Medellín, Colombia.
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis. GBM stem cells (GSCs) are considered to be responsible for tumor progression, therapy resistance and recurrence. The identification of mechanisms that are critical for GSCs maintenance is key to develop better therapy. Previously, we found that GSCs are highly sensitive for endoplasmic reticulum (ER) stress when compared to differentiated counterparts. Since autophagy is one of the known salvage pathways activated by ER stress, here we set out to explore the role of autophagy in the adaptive response of GSCs towards the ER stress inducer Thapsigargin (Tg). Tg induced autophagy activation in all GBM models tested, with no clear correlations with GSC-enriched neurospheres (Nsp) or differentiated counterparts. Tg treatment was accompanied by accumulation of CHOP and PARP cleavage, indicative of apoptosis induction. Effects on autophagic flux were monitored by using the autophagy inhibitor Bafilomycin A1 (BafA1). All GBM models tested showed basal levels of autophagic flux and variable effects of Tg on autophagic flux. Furthermore, Tg-dependent cytotoxicity was potentiated by BafA1 in all three GBM neurospheres, reaching significance only in GG6 Nsp. Transcriptomic analyses revealed down-regulation of several genes active in autophagosome formation in neurospheres versus differentiated GBM cells and may explain a higher dependency on autophagy for cell survival. In conclusion, this preliminary study shows that ER stress induction by Tg leads to autophagy activation in all tested GBM models. GSC-enriched neurospheres seem to be more dependent on this pro-survival route than their differentiated counterparts. Additional work is required to further explore the importance and molecular mechanisms that link ER stress and autophagy in GSCs and to examine therapeutic possibilities.
Introduction
Glioblastoma Multiforme (GBM) is the most common malignancy in the brain, accounting for 45% of malignant primary central nervous system tumors [1,2]. GBM has a very poor prognosis with a 5‐year survival rate of 4–5% and a median survival of around 15 months that is associated with tumor localization and invasive growth, resistance to current therapy and lack of full knowledge of tumor biology resulting in failure of targeted approaches thus far [1,3]. Another explanation for the low survival rates is tumor heterogeneity leading to failure of current treatment regimens to target all cell populations [4]. Particularly, GBM stem cells (GSCs) represent a highly malignant subpopulation that are characterized by their ability to self-renew that is paired with strong tumor-initiating potential [5,6]. Besides, GSCs are considered to be the main drivers for treatment failure and tumor recurrence. A deeper understanding of the mechanisms that maintain GSCs will lead to the identification of novel molecular targets that is critical for developing better therapies for this deadly disease. [7].
The endoplasmic reticulum (ER) is the central intracellular organelle for protein and lipid synthesis and functioning of the secretory pathway. Perturbation of ER homeostasis, such as disturbance of ATP, calcium levels or change in the redox status, can affect protein folding that leads to misfolded protein accumulation and consequently ER stress [8]. This results in activation of an adaptive stress response known as the unfolded protein response (UPR) [9,10]. The ER-resident chaperone, GRP78 or BiP, plays a central role in UPR activation by being recruited to misfolded proteins, thereby releasing the repressive binding to three ER transmembrane proteins, named PERK, IRE1α and ATF6 [11]. These signaling proteins work in concert to mediate the UPR adaptive response that includes the arrest of general protein synthesis, the upregulation of chaperones, and increase of RNA and protein degradation pathways in order to restore protein-homeostasis [12]. However, when damage is overwhelming the UPR sensors signal to induce cell death [13].
Autophagy is another major adaptive response that is essential for maintaining cellular homeostasis. It is a ubiquitous catabolic process that involves the degradation of cytoplasmic components, including whole organelles, via the lysosomal pathway and it is distinct from other degradative pathways such as proteasomal degradation [14]. Different forms of autophagy have been described and here we refer to the most
prominent one, termed macroautophagy. The initials steps in autophagy include the formation and expansion of an isolation membrane, which is also called a phagophore. The edges of the phagophore then fuse to form the autophagosome, a double-membraned vesicle that sequesters the targeted cytoplasmic material. This is followed by fusion of the autophagosome with a lysosome to form an autolysosome where the captured material, together with the inner membrane, is degraded [15]. Autophagy is strongly enhanced in response to various conditions, including nutrient shortage, and the molecular mechanisms driving and regulating this process have been elucidated in detail. The level and rate of autophagy contribute to overall activity and is known as the autophagic flux and can be experimentally explored [16]. For example, microtubule-associated protein light chain 3 (LC3), localized to the autophagosomes, and SQSTM1/ p62 protein, a molecular adaptor between the autophagic machinery and substrates that are destined to be degraded, serve as markers for autophagy activity [17,18].
In cancer, autophagy was found to have two opposite roles. During early transformation autophagy has tumor suppressive activity through the elimination of oncogenic protein substrates, toxic unfolded proteins and damaged organelles. However, at later stages of tumor development it promotes tumorigenesis by stimulating cell survival through autophagy-mediated intracellular recycling of damaged organels or cytotoxic products [19]. Additionally, accumulating evidence suggests that the unique properties of cancer stem cells depend on autophagy [20].
Recent investigations revealed that ER stress in cancer cells can stimulate autophagy as a survival mechanism [21]. Different molecular mechanisms have been found that link the UPR to autophagy. For example, the IRE1/ XBP1 branch can induce the autophagy regulator Beclin-1; PERK-dependent ATF4 expression can induce the autophagy-related genes ATG5 and ATG12 and ATF6 can activate HSPA5, Beclin-1 or
CaMKK leading to suppression of the autophagy inhibitor mTOR [8,21]. In vitro studies
with chemical modulators of autophagy like cyclosporine, minocycline or curcumin have demonstrated that ER stress-dependent autophagy activation contributes the survival of GBM cells [22-25]. However, the role of ER stress-associated autophagy in the maintenance and survival of GSCs has been not fully addressed. Therefore in the current preliminary study we set out to investigate the activation of autophagy by ER stress and its contribution to cytotoxicity in our GSC-enriched GBM neurospheres and their differentiated counterparts.
The human GBM cell lines GG6, GG16 and GSC23 were patient-derived from GBM primary material. GBM neurospheres (Nsp) and differentiated (Diff) cells were cultured in neural stem cell medium (NSM) and serum (10% FCS) supplemented differentiation medium respectively, as previously described [26,27].
Cells were treated with the ER sarco/endoplasmic reticulum calcium ATPase (SERCA) pump inhibitor Thapsigargin (Tg) (Sigma-Aldrich, Zwijndrecht, the Netherlands) using the IC50 for each cell line and conditions according to previous results (Chapter 3). 5 nM of Bafilomycin A1 (BafA1) (Sigma-Aldrich, Zwijndrecht, the Netherlands) was used for 24 h as an autophagy inhibitor to determine autophagic flux. Untreated cells or cells treated with DMSO solvent were used in parallel as controls.
Cells were seeded and after 24 h pre-culturing exposed to Tg and/or BafA1 (in case of combined use administrated at the same time) for another 24 h. Cells were lysed with M-PER mammalian protein extraction agent (Thermo Fisher Scientific, Bleiswijk, the Netherlands) supplemented with 1% protease inhibitor cocktail (Thermo Fisher Scientific, Bleiswijk, the Netherlands) and 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific, Bleiswijk, the Netherlands). Protein concentration measurements were performed using Bradford Protein Assay (BioRad, Veenendaal, the Netherlands) according to the manufacturer’s instructions. Subsequent Western Blot procedure was performed as described previously [27]. The membranes were incubated overnight with the indicated primary antibody. Primary antibodies used were rabbit polyclonal against SQSTM1/p62 (#8025), rabbit polyclonal against LC3 I/II (#4108), mouse monoclonal against CHOP (#2895),rabbit monoclonal against PARP (#9532) (all from Cell Signaling Technology, Bioké, Leiden, the Netherlands), rabbit polyclonal against BiP/GRP78 (ab21685) (Abcam, ITK diagnostics BV, Uithoorn, Netherlands) and mouse monoclonal anti-β-actin (69100) (ICN Biomedicals, Zoetermeer, the
Materials and Methods
Cell Culture and treatmentsTo measure autophagic vacuoles and monitor autophagic flux we used the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Bruxelles, Belgium). Cells were pre-cultured in a 24-well plate at a density of 2 x 105 cells/well for 24 h. After treatment with Tg
and/or BafA1 (in case of combined use administrated at the same time), freshly diluted Cyto-ID green detection reagent and Hoechst were added to the culture medium and subsequently incubated for 30 min at 37°C. Excess dye was removed by one or two washes using assay buffer. Fluorescent images of each staining were visualized by fluorescence microscopy (Leica DM-6000 Microscope; Wetzlar, Germany) and images of each condition were captured. Three independent experiments were performed.
Cells were seeded in triplicate in 96-well plates at a cell density of 1 x 104 cells/well
and incubated for 24 h prior to treatment with the drugs Tg and/or BafA1 at the same time at indicated concentrations and time periods. After treatment, cell viability was determined using MTS assay by incubation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium solution (MTS) according to manufacturer’s instructions (Promega Corporation, Leiden, the Netherlands). Cell viability was determined by measuring the absorption at 490 nm on a Microplate reader (BioRad, Veenendaal, the Netherlands). Three independent assays were performed in triplicate.
Cells were treated as indicated and RNA was isolated from cell pellets using TRIzol®
Autophagosome detection by fluorescent microscopy
Cell Viability Assay
RNA isolation
Netherlands). Three independent experiments were performed except for figure 1 showing the preliminary results of one experiment.
Reagent (Life Technologies, Thermo Fisher Scientific, Bleiswijk, the Netherlands) following the manufacturer’s protocol. RNA quantification and cDNA synthesis was performed as previously described [28]. Three independent experiments were performed.
Illumina Next Generation Sequencing was performed by the Genome Analysis Facility (GAF), Genomics Coordination Centre (GCC) at University Medical Centre Groningen (Groningen, the Netherlands). Initial quality check and RNA quantification of the samples was performed by capillary electrophoresis using the LabChip GX (Perkin Elmer, Groningen, the Netherlands). Non-degraded RNA-samples were selected for subsequent sequencing analysis. Sequence libraries were generated using the TruSeq RNA sample preparation kits (Illumina) using the Sciclone NGS Liquid Handler (Perkin Elmer, Groningen, the Netherlands). In case of contamination of adapter duplexes an extra purification of the libraries was performed with the automated agarose gel separation system Labchip XT (PerkinElmer, Groningen, the Netherlands). The obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2500 using default parameters (single read 1x50bp or Paired End 2 x 100 bp) in pools of multiple samples. Sequenced reads were trimmed and subsequently aligned to build b37 human reference genome using HISAT2 0.1.5 [29] and SAMtools 1.2 [30] allowing for two mismatches. Gene level quantification was done using HTSeq/0.6.1p1 [31] using --mode=union--stranded=no. Ensembl v75 was used as reference for gene annotation. Overall genes with less than 40 reads were kept out of the analysis. Then reads counts were normalized using trimmed mean of the M-values method. Differential expression (DE) analysis between conditions was done using the DESeq2 package [32] for R (http://www.r-project.org/). Each DE analysis was performed using paired samples and including library size as covariate. Genes that had an adjusted p-value ≤ 0.05, and a Log2 Fold Change more or less than 1 were define as significant differentially expressed genes (DEG)
Statistical analysis of cytotoxicity experiments was performed using double sided, paired or unpaired (depending on conditions) Student t-test. Statistical significance was considered at p < 0.05. Statistics used for the sequence analysis are described in the corresponding section.
In order to study the contribution of autophagy to the adaptive response of Tg-treated GBM cells, we determined levels of LC3-I/II and p62 expression as markers for autophagy in GBM neurospheres and differentiated counterparts. LC3 is conjugated to phosphatidylethanolamine during autophagy to form LC3-II. LC3-II is subsequently recruited to autophagosome membranes and is degraded in autolysosomes. The amount of LC3-II usually correlates well with the number of autophagosomes and thus autophagy activity [16]. Cells were treated for 1, 4, 8, 12 and 24 h with Tg at the previously determined IC50 concentrations (see Chapter 3). Activation of the UPR was confirmed by a time-dependent increase in BiP/GRP78 expression in the GG6, GG16 and GSC23 models (Figure 1). LC3-I/II expression was already detected in untreated cells and Tg generally resulted in increased expression at later time-points of exposure showing little differences between neurospheres and differentiated cells.
P62 binds to polyubiquitinated proteins and interacts directly with LC3 to degrade the cargo in autolysosomes. A decrease in p62 reflects autophagy activation, whereas accumulation of p62 indicates autophagic inhibition. It should be noted that p62 is also degraded by the ubiquitin-proteasomes system [18,33]. A small decrease in p62 was seen in GG6 and GSC23 Nsp. In GG16 Nsp, an initial clear decrease in p62 was followed by an increase, possibly indicating enhanced autophagy at early time points. In the differentiated counterparts p62 levels remained mostly unaltered with increases at late time-points particularly in GSC23.
The triggering of apoptosis was monitored by assessing PARP (Poly (ADP-ribose)
Statistical analysis
LC3-I/II and p62 expression in Tg-treated GBM cells
Figure 1: Tg induced autophagy in GBM neurospheres and differentiated cells. Immunoblots
showing BiP/GRP78, LC3-II conversion, p62 levels, CHOP expression and cleavage of PARP over time upon Tg-induced ER stress activation. Cells were treated with corresponding IC50 Tg doses (n=1).
polymerase) cleavage. Weak PARP cleavage was already detected at early time points of Tg exposure; strongest cleavage was detected at 24 h Tg treatment. GG16 Diff and GSC23 Diff cells showed less PARP cleavage compared to the Nsp, whereas in GG6 cells this seemed to be the opposite although cleavage reduced at 24h post-treatment. In parallel, accumulation of apoptosis-inducing CHOP (CCAAT-enhancer-binding protein homologous protein) was detected in both neurospheres and differentiated cells at early time points, although somewhat stronger in neurospheres.
Next, autophagosome formation was determined using specific staining with a fluorescent dye in both GBM neurospheres and differentiated cells upon exposure to Tg. Fluorescence microscopy revealed Tg-induced autophagosome formation in all GBM models tested, although the number of positive cells and fluorescent intensity varied. Treatment with Tg for 24h resulted in strong autophagosome accumulation in GG6 Nsp, while weaker staining was seen in GG16 and GSC23 Nsp (Figure 2A). Tg-treated differentiated counterparts also displayed variable autophagosome accumulation, with lowest number of positive cells detected specially in GG6.
Autophagy is a highly dynamic process and the rate between autophagosome formation, lysosomal fusion (autolysosome) and degradation, known as the autophagic flux, is important for overall autophagic activity. This can be monitored by blocking lysosomal degradation with the pharmacological inhibitor BafA1. In all three GBM models, in both neurospheres and differentiated cells, treatment with BafA1 alone increased the amount of autophagosomes compared to untreated control cells, indicating basal levels of autophagic flux in the absence of ER stress aggravation. Combined Tg and BafA1 treatment showed cell- and differentiation status-dependent variable effects on autophagosome accumulation (Figure 2A). Overall, in Nsp cells combined Tg/BafA1 treatment showed similar levels of autophagosome accumulation compared to BafA1 treatment alone, but lower levels compared to Tg alone. (Figure 2A). In the differentiated cells combined treatment showed comparable autophagosome accumulation compared to drug-only treatments. Together, these qualitative data indicate that Tg activates autophagy in all tested GBM models and are suggestive for a somewhat stronger activation in neurospheres.
The autophagic flux was also studied by means of detecting LC3I/II conversion and p62 levels on Western blots. After 24 h Tg treatment, increased LC3-II expression
Autophagosome formation and autophagic flux in Tg exposed GBM cells
Overall, evidence for variable levels of autophagy activation during time was obtained after Tg exposure in all tested GBM cells with no clear correlation with the GBM differentiation status, which was accompanied by accumulation of CHOP and PARP cleavage, indicative of apoptosis activation.
was seen in GG6 and GG16 cells, but only marginal in GSC23 cells (Figure 2B), in both neurospheres and differentiated cells. BafA1 alone resulted in even stronger increases of LC3-II levels, indicative of basal autophagic flux in all models in the absence of exogenous ER stress. In GSC23 cells combined Tg/BafA1 exposure did not affect LC3-I/II levels when compared to BafA1- or Tg-alone treated cells, whereas levels appeared lower in GG6 cells and were elevated in GG16 cells. Increases in p62 expression mostly followed LC3-II expression patterns.
GG6 GG16 GSC23 Tg [IC50µM] - + - + - + BafA1[5nM] -+ -+ Nsp Diff Nsp Diff β-actin LC3 I/II p62 β-actin LC3 I/II p62 BafA1 [5nM] Tg [IC50µM] - - + + - + - + GG6 - - + + - + - + GG16 - - + + - + - + GSC23 A B
Figure 2: The effect of Tg on autophagic flux in the GBM models. (A) GBM neurospheres
(Nsp) and differentiated (Diff) cells were incubated with IC50 Tg dose for 24 h in the presence or absence of 5 nM BafA1 and stained with Cyto-ID dye (green) to detect (B) Representative immunoblots showing LC3-II conversion and p62 levels under similar Tg and BafA1 exposure as under A.
0 20 40 60 80 100 ** ** * Tg [3µM] - - + + BafA1 [5nM] - + - + 0 20 40 60 80 100 * * Tg [6µM] - - + + BafA1 [5nM] - + - + 0 20 40 60 80 100 ** ** Tg [6µM] - - + + BafA1 [5nM] - + - + 0 20 40 60 80 100 ** ** Tg [5µM] - - + + BafA1 [5nM] - + - + 0 20 40 60 80 100 * * Tg [10µM] - - + + BafA1 [5nM] - + - + A B Ce ll via bilit y (% of c ont rol ) 0 20 40 60 80 100 * * Tg [6µM] - - + + BafA1 [5nM] - + - + Ce ll via bilit y (% of c ont rol ) Ce ll via bilit y (% of c ont rol ) Ce ll via bilit y (% of c ont rol ) Ce ll via bilit y (% of c ont rol ) Ce ll via bilit y (% of c ont rol ) Nsp Diff GG6 GG16 GSC23
The effect of autophagy inhibition on Tg-induced cytotoxicity
The effect of Tg-induced autophagy activation on GBM cell viability was examined. BafA1 treatment alone for 24 h did not affect cell viability in GBM neurospheres and differentiated cells. However, Tg-dependent cytotoxicity was potentiated by BafA1 in all three GBM Nsp, but reached only significance in GG6 Nsp cells (Figure 3A). Notably, autophagy appeared not to contribute to the survival of Tg exposed differentiated cells (Figure 3B).
Together, these findings suggest a Tg-dependent increase of autophagic flux in GG16 Nsp/Diff, whereas no clear differences in autophagic flux after Tg exposure could be appreciated in the other models.
Figure 3: Effect of autophagy inhibition on Tg-induced cytotoxicity in the GBM models.
Histograms of cell viability (%) assessed by MTS assay. GBM neurospheres (Nsp) (A) and GBM differentiated (Diff) (B) cells were exposed to corresponding IC50 Tg doses for 24 h in the presence or absence of BafA1. Error bars indicate standard deviations. *P-value<0.05 and **P-value<0.001.
To obtain more insight in possible differences in autophagy activity in neurospheres and differentiated GBM cells, we compared transcript levels of genes related to the autophagy pathway in GG16 and GSC23 cells. Using RNAseq datasets, differential expression patterns of the autophagy-related genes were detected in neurospheres compared to differentiated cells (Figure 4A). We selected genes with a 2-fold or more change in expression subdivided in two categories, ‘autophagy machinery components’ (Figure 4B) and ‘autophagy regulatory genes’ (Figure 4C). In GG16 Nsp the early autophagic stimulatory genes microtubule associated protein 1 light chain 3 alpha (MAP1LC3A, also known as LC3 or ATG8) and ATG4C were down-regulated, the Niemann-Pick type C1 (NPC1) involved in autophagosome maturation and cholesterol metabolism were up-regulated. The expression of apoptotic regulator BH3 interacting domain death agonist (BID) was reduced. In GSC23 Nsp, MAP1LC3A, ATG4A and
FAM134B that are all linked with autophagosome formation were downregulated,
whereas ATG2B stimulating autophagosome formation went up. Co-regulators of autophagy and apoptosis Apolipoprotein L1 (APOL1), Tumor necrosis factor super family member 10 (TNFSF10/TRAIL), Cathepsin S (CTSS), Bcl-2 associated death promoter (BAD), Cathepsin D (CTSD), Bcl-2 associated X (BAX), FAS, and Lysosomal acid α-glucosidase (GAA) were significantly down-regulated in GSC23 Nsp compared to differentiated counterparts and Unc-51 like autophagy activating kinase (ULK2), B-cell lymphoma 2 (Bcl2), Bcl2-interacting protein 3 (BNIP3) and EIF2AK3 (PERK) were up-regulated in GSC23 Nsp. Overall it appears that several genes involved in the early formation of autophagosomes are decreased in neurospheres and that genes regulating apoptosis are decreased in differentiated cells, particularly in GSC23 Diff, which may affect the status of the autophagy machinery.
Differences in autophagy-related gene transcript levels in neurospheres versus differentiated GBM cells
Autophagy Machinery Components Nsp Vs. Diff Regulation Autophagy Nsp Vs. Diff A B GSC23 GG16 GSC23 GSC23 MAP 1LC3A FAM13 4B ATG4AATG2B -4 -2 0 2 Log 2 Fol d Cha ng e APOL 1 TNFSF1 0 CTSSBADCTSDBAX FASGA A ULK2BCL2 EIF2 AKBNIP3 3 -4 -2 0 2 Log 2 Fol d Cha ng e GG16 C BID -4 -2 0 2 Log 2 Fol d C ha ng e GG16 MAP1LC3AATG4 C NPC1 -4 -2 0 2 Log 2 Fol d C ha ng e
Figure 4: Transcriptomic differences of autophagy related genes in GBM neurospheres and differentiated cells. (A) Autophagy-related transcripts were compared in GG16 and
GSC23 neurospheres versus their differentiated counterparts. Genes that have an adjusted p-value ≤ 0.05 are depicted in heatmaps as differentially expressed. Color scale represents z-scores. Significantly differentially expressed genes that have an adjusted p-value ≤ 0.05 and a Log2 Fold Change > 1 related with (B) autophagy machinery components or (C) regulation autophagy genes are depicted.
Discussion
In this study, we show that ER stress induced by Tg leads to activation of autophagy in all tested GBM models based on the overall found increases in LC3-II conversion, p62 reduction and accumulation of autophagosomes. Results of our study are suggestive for a higher dependency of GSC-enriched neurospheres on autophagy, however, due to cell-dependent variability in Tg-induced changes including on autophagic flux it is too early to draw firm conclusions as yet.
Autophagy is integrated with proteasomal degradation and the UPR to govern cell fate through restoration of cellular homeostasis or default into the apoptotic cell death pathway [34]. Accordingly, in some circumstances autophagy is involved in the prevention of cell death by removing accumulated polyubiquitinated proteins and aggregates. Our results indicate that autophagy also acts to prevent cell death particularly in Tg-treated neurospheres, since inhibition of autophagy with BafA1 enhanced Tg cytotoxicity in these cells. Numerous reports have shown that blocking autophagy can enhance ER stress-induced cell death, also in GBM U87 cells monolayer model [35,36]. Several properties that are also shared by GSCs, such as self-renewal, pluripotency and differentiation, were demonstrated to rely on autophagy activity [37,38]. This appears in line with the increased sensitivity to autophagy inhibition that we observed in neurospheres cells, although the effect on GSC self-renewal and differentiation remains to be examined.
In this study, we used Tg as an ER stress inducer, however, several reports have indicated that Tg may affect autophagy via different ways. Tg, being an irreversible non-competitive inhibitor of the SERCA pump, raises intracellular calcium (Ca2+)
concentrations by blocking the ability of the cell to pump Ca2+ into the ER [39]. The role
of intracellular Ca2+ as one of the regulators of autophagy process is well established
[40,41]. Sakaki et al. showed that Tg-induced autophagy was still present in UPR-deficient cells, suggesting a direct involvement of Ca2+ in the induction of autophagy
[42]. Nevertheless, the precise role of Ca2+ in Tg-blocked autophagy is still unknown.
To better understand the role of autophagy and ER stress in the cytotoxic response in GSCs, additional ER stress inducers should be employed (e.g. tunicamycin, an inhibitor of N-linked glycosylation) to further determine the role of UPR-activated autophagy in cell survival and self-renewal. The use of genetic
knock-down/knock-out approaches of autophagy-related genes (e.g. ATG5, ATG7) and UPR regulatory proteins could bring further insight in how autophagy and ER stress act in concert to maintain GSCs.
Lastly, in order to examine possible differences in autophagic activity between neurospheres and differentiated cells transcriptional profiles were compared. We found that MAP1LC3A, ATG4C, ATG4A and FAM134B were downregulated in neurospheres. These are linked with autophagosome structure [43], formation [44] and the recruitment of autophagosomes to the ER [45], suggestive of reduced autophagosome formation, although care should be taken drawing correlations between transcript levels and functional activity. On the other hand genes regulating apoptosis were downregulated mostly in GSC23 Nsp and may shift the balance to autophagy. Together, a higher dependency of neurospheres on autophagy is congruent with a report by Ciechomska et al. [20] who found that GSCs-enriched spheres originating from glioma cell lines and GBM patient-derived cultures express lower levels of autophagy-related genes compared to differentiated counterparts, also suggesting reduced autophagy in GSCs. It may be speculated that ER stress-induced activation of autophagy will have a larger impact in cells, in this case GSCs, with lower basal levels of autophagy.
Taken together, we demonstrated that autophagy is activated to variable extents in response to Tg-induced ER stress in neurospheres and differentiated counterparts and that particularly neurospheres may be dependent on its pro-survival activity. However, our current study is preliminary and explorative in nature, providing a basis for further studies on the importance and molecular mechanisms that link ER stress and autophagy in GSCs. Simultaneously modulation of these pathways may provide a new therapeutic approach.
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