Engagement of cellular prion protein with the co-chaperone Hsp70/90 organizing protein regulates the proliferation of glioblastoma stem-like cells
© The Author(s). 2017
Received: 15 October 2016
Accepted: 21 February 2017
Published: 17 April 2017
Glioblastoma (GBM), a highly aggressive brain tumor, contains a subpopulation of glioblastoma stem-like cells (GSCs) that play roles in tumor maintenance, invasion, and therapeutic resistance. GSCs are therefore a promising target for GBM treatment. Our group identified the cellular prion protein (PrPC) and its partner, the co-chaperone Hsp70/90 organizing protein (HOP), as potential target candidates due to their role in GBM tumorigenesis and in neural stem cell maintenance.
GSCs expressing different levels of PrPC were cultured as neurospheres with growth factors, and characterized with stem cells markers and adhesion molecules markers through immunofluorescence and flow cytometry. We than evaluated GSC self-renewal and proliferation by clonal density assays and BrdU incorporation, respectively, in front of recombinant HOP treatment, combined or not with a HOP peptide which mimics the PrPC binding site. Stable silencing of HOP was also performed in parental and/or PrPC-depleted cell populations, and proliferation in vitro and tumor growth in vivo were evaluated. Migration assays were performed on laminin-1 pre-coated glass.
We observed that, when GBM cells are cultured as neurospheres, they express specific stemness markers such as CD133, CD15, Oct4, and SOX2; PrPC is upregulated compared to monolayer culture and co-localizes with CD133. PrPC silencing downregulates the expression of molecules associated with cancer stem cells, upregulates markers of cell differentiation and affects GSC self-renewal, pointing to a pivotal role for PrPC in the maintenance of GSCs. Exogenous HOP treatment increases proliferation and self-renewal of GSCs in a PrPC-dependent manner while HOP knockdown disturbs the proliferation process. In vivo, PrPC and/or HOP knockdown potently inhibits the growth of subcutaneously implanted glioblastoma cells. In addition, disruption of the PrPC-HOP complex by a HOP peptide, which mimics the PrPC binding site, affects GSC self-renewal and proliferation indicating that the HOP-PrPC complex is required for GSC stemness. Furthermore, PrPC-depleted GSCs downregulate cell adhesion-related proteins and impair cell migration indicating a putative role for PrPC in the cell surface stability of cell adhesion molecules and GBM cell invasiveness, respectively.
In conclusion, our results show that the modulation of HOP-PrPC engagement or the decrease of PrPC and HOP expression may represent a potential therapeutic intervention in GBM, regulating glioblastoma stem-like cell self-renewal, proliferation, and migration.
KeywordsCellular prion protein Hsp70/90 organizing protein Glioblastoma Stem cells Proliferation
Glioblastoma (GBM) is the most common and aggressive type of central nervous system (CNS) tumor and is one of the most lethal human cancers . This aggressive behavior has been attributed to a highly proliferative subset of cells called glioblastoma stem-like cells (GSCs) that contribute to tumor initiation and therapeutic resistance . These cells are thought to be responsible for maintaining GBM tumors after therapy and repopulating them after total resection . In addition, they are involved in tumor angiogenesis  and immune evasion , promoting tumor progression.
The cellular prion protein (PrPC) and the Hsp70/Hsp90 organizing protein (HOP) modulate several stem cell functions. PrPC regulates proliferation and self-renewal of neural stem cells (NSCs) , while HOP participates in the maintenance of pluripotent stem cells . Previous data from our group show that PrPC modulates NSC proliferation and self-renewal through its interaction with HOP . Both PrPC and HOP also modulate tumorigenesis, affecting the progression and maintenance of different types of cancers . PrPC associates with a poor clinical outcome and survival in pancreatic ductal adenocarcinoma and melanoma [9, 10], and with invasion and metastasis in gastric and breast cancers [8, 11, 12]. Depleting PrPC inhibits growth, promotes programmed cell death in gliomas , and sensitizes tumor cells to cytotoxic drugs . Likewise, HOP expression correlates with tumor progression , and is associated with proliferation [16, 17], invasion , and poor patient prognosis , and HOP has been described as an important regulator of tumor maintenance [19–21]. Recent findings show that HOP-PrPC binding modulates migration and invasion of colorectal cancer cells . Erlich et al.  reported that the interaction of PrPC with HOP modulates proliferation in glioma cell lines, and data from our group showed that higher expression of both proteins is correlated with greater tumor proliferation and lower survival in patients with GBM . In addition, blocking the HOP-PrPC complex decreases tumor growth and increases survival in an animal model , making it a potential target for GBM therapy.
While the HOP-PrPC complex participates both in GBM tumorigenesis and in the maintenance of NSCs—which are believed to originate GSCs—their role in GSC biology is still unclear. Therefore, this study aimed to elucidate the role of the HOP-PrPC complex in the regulation of GSCs by downregulating the expression of PrPC in a human GBM cell line cultured as GSCs and by testing how proliferation and self-renewal properties were affected in the presence of exogenous HOP; we also wished to evaluate the therapeutic potential of targeting this complex using synthetic peptides in attempt to alter GSC biology. Additionally, we investigated whether PrPC may be used as a novel biomarker for GBM by studying its role in GSC stemness.
Proteins and peptides
Mouse recombinant HOP was purified as previously described . Human pepHOP230–245 (ELGNDAYKKKDFDTAL) and C-terminal pepHOP422–437 (GCKTVDLKPDWGKGYS) peptides were synthesized by GenScript (Piscataway, NJ, USA).
The human U87 and U251 glioblastoma cell lines (ATCC) were cultured in DMEM-F12, supplemented with B27 (Cat No. 17504-044; Gibco, Gaithersburg, MD, USA; 1:50) in the presence of 20 ng/ml epidermal growth factor (EGF; Cat No. E4127; Sigma Aldrich, St. Louis, MO, USA) and basic fibroblast growth factor (bFGF; Cat No. F0291; Sigma Aldrich) at 37 °C under 5% CO2 to form neurospheres. The medium was replaced every 2 days. After 1 week, cells were treated with 0.25% trypsin (Cat No. 25200-056; Gibco) in HBSS (Cat No. 14170-112; Gibco) for 5 min at 37 °C. Trypsin was washed out and the cells were mechanically dissociated and plated for distinct assays.
shRNA-PrP2: 5′- caccagaacaacttcgtgcacgactcgaaaagtcgtgcacgaagttgttc-3′
Stable silencing of HOP was performed using MISSION® shRNA (Sigma Aldrich) according to the manufacturer’s instructions using the following sequences: TRC 2.0; NM_006819 – Mission – SIGMA/TRCN0000243096 and TRCN0000243099.
CRISPR/Cas9 for PrPC knockout
The human PrPC gene sequence (NM_000311.3) was used to design the guide RNA using the Optimized CRISPR Design (http://crispr.mit.edu/). gRNA sequences selected were: Hu PrPC (Top1) CACCGgctgggggcagccgatacccg/Hu PrPC (Bottom1) AAACcgggtatcggctgcccccagcC. The gRNAs were phosphorylated, annealed, and cloned into px330-U6-GFP vector (Addgene) according to the Addgene website instructions. The construct was sequenced, transfected into the U251 cell line with lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions, and the clones were isolated by serial dilution.
Flow cytometry analysis
Cells (106) were dissociated, washed twice with phosphate-buffered saline (PBS), and incubated with anti-PrPC , anti-CD133 (Cat No. 130-090-852; Miltenyi, Auburn, CA, USA), CD15 (Cat No. FCMAB182F; Millipore, Temecula, CA, USA), anti-E-cadherin (Cat No. 610181; BD Bioscience, San Diego, CA, USA), and anti-integrin α6 (Cat No. ab97760; Abcam, Cambridge, UK) antibodies, all at 1:50 dilution in 0.5% bovine serum albumin (BSA) in PBS for 60 min at 4 °C. After washing, samples were incubated with anti-mouse IgG Alexa-488/PE (Cat No. A21200; Invitrogen, Carlsbad, CA, USA; 1:200) or with anti-rabbit IgG Alexa-488/405 (Cat No. A21441; Invitrogen; 1:200) antibodies for 60 min at 4 °C. For PrPC internalization assays, cells were pre-incubated for 40 min in 250 μM solution of CuSO4 in PBS plus 5% BSA at 37 °C, followed by antibody incubation. Only secondary staining was used for the negative control. Cells were analyzed by flow cytometry for forward scatter, side scatter, and fluorescence (FACSCanto II; BD Biosciences).
For the analysis of neurosphere protein extracts, cells were cultured overnight (2 × 105 cells/well), starved for 24 h, treated with serum or recombinant HOP for 15 min, and washed with cold PBS. Protein extracts were prepared in RIPA buffer with protease/phosphatase inhibitors, centrifuged (10,000 × g), and then loaded (5 μg) onto a 10% SDS-PAGE gel, followed by immunoblotting with polyclonal anti-HOP (1:10,000) , anti-phospho Erk1/2 (Cat No. 4370S; Cell Signaling, Danvers, MA, USA; 1:4000), anti-Erk1/2 (Cat No. 4695S; Cell Signaling; 1:4000) or anti-PrPC . Anti-GAPDH (Cat No. G9545; Sigma Aldrich) or anti-actin antibodies (Cat No. A2103; Sigma Aldrich) were used as protein loading controls.
For PrPC and Oct4 staining, whole neurospheres were harvested, fixed in 4% paraformaldehyde, and paraffin-embedded. For Ki67 experiments, xenografts (Balb/c nude mice) were resected, fixed in 4% paraformaldehyde, and paraffin-embedded. Slides with 3-μm sections were prepared for immunofluorescence. Sections were incubated in xylol at 60 °C for paraffin removal and immersed in citrate buffer, pH 6.0, for 1 h at 96 °C for antigen retrieval. Sections were then blocked for 1 h at room temperature (RT) in 5% BSA in PBS and incubated overnight at RT with anti-PrPC (1:50)  or anti-Oct4 (Cat No. 2840S; Cell signaling; 1:50) in 1% BSA in PBS. After washing, slides were incubated for 1 h at RT with anti-mouse Alexa-488 (Cat No. A21202; Invitrogen; 1:1000) or anti-rabbit Alexa-546 (Cat No. A10040; Invitrogen; 1:1000), and stained with TO-PRO (Cat No. T3605; Molecular Probes, Eugene, OR, USA) for nuclei. For other markers, whole neurospheres were harvested, plated on coverslips previously treated with poly-l-lysine and fixed in 4% paraformaldehyde. Coverslips were blocked for 1 h at RT with 5% BSA plus 0.3% triton in PBS. Coverslips were incubated overnight at RT with anti-PrPC (1:100) and anti-HOP (1:100) , anti-nestin (Cat No. N5413; Sigma Aldrich; 1:100), anti-Musashi1 (Cat No. 5663P; Cell Signaling; 1:100), anti-Sox2 (Cat No. ab75485; Abcam; 1:100), anti-βIII tubulin (Cat No. 5568S; Cell Signaling; 1:100), anti-β catenin (Cat No. ab32572; Abcam; 1:100), anti-CD133 (Cat No. MAB4399; Millipore; 1:100), anti-GFAP (Cat No. Z0334; Dako, Cambridge, UK; 1:100) or anti-E-cadherin (Cat No. 610181; BD Bioscience; 1:100) in 1% BSA 0.1% triton in PBS. After washing, coverslips were incubated for 1 h at RT with anti-mouse Alexa-488 (Cat No. A21202; Invitrogen; 1:1000) or anti-rabbit Alexa-546 (Cat No. A10040; Invitrogen; 1:1000), and stained with TO-PRO (Cat No. T3605; Molecular Probes) or DAPI (Cat No. D1306; Invitrogen) for nuclei. Cells were imaged on a Leica TCS SP2 II laser scanning confocal system.
Cell proliferation assay
Whole neurospheres were harvested and plated on coverslips previously treated with poly-l-lysine in DMEM-F12 supplemented with B27 at 37 °C. Control cells were treated only with the growth factors (20 ng/ml) EGF (Cat No. E4127; Sigma Aldrich) and bFGF (Cat No. F0291; Sigma Aldrich), and for HOP treatments cells were treated with growth factors and recombinant HOP and/or HOP peptides (1 μM) for 24 h. Cells received a 60-μM BrdU pulse 3 h prior to fixation in 4% paraformaldehyde. Fixed cells were treated with HCl 2 N for 30 min, washed with borate buffer, and permeabilized with 0.3% Triton X-100 in PBS for 15 min. Cells were blocked in 0.3% Triton 5% BSA in PBS for 1 h and stained with biotin-conjugated anti-BrdU (Cat No. MAB3262B; Millipore; 1:100), Strepta-AlexaFluor-546 (Cat No. S11225; Molecular Probes; 1:500), and DAPI (Cat No. D1306; Invitrogen; 1:500) for nuclei. Images were taken from at least four microscopic fields (Zeiss AxioVertA1) for each duplicate per treatment and analyzed on ImageJ software (NIH). The percentage of BrdU-positive nuclei in the total number of nuclei (DAPI) was calculated. HOP-silenced populations were plated 24 h prior to performing the transfection and proliferation assays (as described above). For colorimetric assays, cells were treated with growth factors and recombinant HOP and/or HOP peptides (1 μM) for 24 h and the Cell Proliferation ELISA BrdU kit (Cat. No. 11647229001; Roche, Indianapolis, IN, USA) was utilized according to the manufacturer’s instructions.
Clonal density assay
Neurospheres were treated with 0.25% trypsin (Cat No. 25200-056; Gibco) in HBSS (Cat No. 14170-112; Gibco) for 20 min at 37 °C. Trypsin was washed out and cells were mechanically dissociated. In each well (96-well plate), 200 cells were plated in triplicates per condition (control or treatment with 500nM recombinant HOP and/or HOP peptides). Cells were treated every 48 h for 1 week. Images were acquired through light microscopy (Zeiss PrimoVert) and the number and size of neurospheres were evaluated and compared between conditions with the ZEN software (Zeiss). The optimal concentration of HOP for self-renewal or proliferation assays was chosen based on previous experiments of dose-response curve (data not shown) or according to previous data , respectively.
Neurospheres were plated on coverslips previously treated with poly-l-lysine and laminin-1 (5 μg/ml) in DMEM-F12 supplemented with B27, and the growth factors EGF (Cat No. E4127; Sigma Aldrich) and bFGF (Cat No. F0291; Sigma Aldrich) and 2% fetal bovine serum (FBS; Lot No. 003/14; Vitrocell, Campinas, Spain), and cultured for 24 h at 37 °C. Images were acquired through light microscopy (ZeissPrimoVert) and the halo of migration was compared to neurosphere radius to evaluate cell migration using ZEN software (Zeiss). For cell scratch assays, neurospheres were dissociated and plated 0.2 × 106 per well (six-well plate), the scratch was performed, and images were acquired at 0 h and 24 h after the scratch. Images of three experimental replicates were acquired using Zeiss PrimoVert microscope and the distance of each scratch closure after 24 h was measured by comparing with the images at time 0 h using ZEN software (Zeiss).
In vivo tumor growth
Institutional guidelines for animal welfare were followed and the study approved by the Animal Ethics Committee of the Institute of Biomedical Sciences/University of Sao Paulo (book 03, page 15, protocol number 002 of 04/03/2014). Neurospheres (1 × 106) as single cells were injected subcutaneously into the flank of female Balb/C nude mice (12 weeks old) in PBS. Tumor growth was measured every 2 days and euthanasia by CO2 saturation was performed on day 10 after tumor detection. Tumors were resected and fixed in paraformaldehyde 4%.
One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used for multiple comparisons. A p value <0.05 was considered statistically significant. The non-parametric Student’s t test was also used in migration assays. Mean values represent at least three independent data sets; error bars represent standard errors of the mean (SEM).
Characterization of neurosphere culture from GBM cell line
Effect of PrPC modulation on the expression of stem-cell markers
Due to their roles in GBM and NSCs, PrPC and HOP were proposed to modulate essential functions in GSC biology. To better understand these functions, an shRNA lentivirus system was used in the U87 cell line to silence PrPC to intermediary (shRNA-PrP1) and low (shRNA-PrP2) levels (Fig. 1e and f). The CRISPR/Cas9 technique was used to generate PrPC knockout U251 cells (Fig. 1g). U251 clone 2 presented clear depletion of PrPC expression (PrPKO) and was selected to perform functional experiments (Fig. 1g and h). Both monolayer populations (shRNA-PrP1and shRNA-PrP2), previously used in , and also PrPKO were cultured as neurospheres.
The stem cell marker CD133 expression is decreased in the U87 PrP2 population compared to the parental population (Fig. 2e), and Sox2 had higher expression in the parental population than in the PrP2 population (Fig. 2f). Musashi-1, on the other hand, presented different cellular locations: nuclei for parental population and cytoplasm for PrP2 (Fig. 2f). No significant difference in expression of the neural precursor marker nestin was observed between populations (Fig. 2f). Cellular differentiation markers, such as GFAP and βIII-tubulin, were highly expressed in PrP2 cells compared to parental neurospheres after serum stimulation (Fig. 2g). These findings reveal a potential role for PrPC in the regulation of the GSC multipotent status.
The PrPC-HOP complex modulates GSC proliferation
We evaluated the effect of exogenous (recombinant) HOP and synthetic peptides (pepHOP230–245 which mimics the PrPC binding site, and peptide pepHOP422–437 used as the control) on the proliferation of parental, PrP1, and PrP2 neurospheres. After 24 h of treatment, there was a significant increase in proliferation of parental and of PrP1 neurospheres treated with HOP, compared to PrPC silenced cells (PrP2) (Fig. 3g). On the other hand, populations pre-treated with pepHOP230–245 were unable to proliferate in the presence of HOP (Fig. 3g), suggesting that pepHOP230–245 is able to block the PrPC-HOP interaction and impair GSC proliferation. Moreover, we observed that HOP activates the Erk1/2 pathway only in neurosphere cultures expressing high levels of PrPC (Fig. 3h and i). PrPC-depleted cells have increased basal levels of phospho-Erk1/2 (Fig. 3h and i), corroborating our previous data in primary cultures from PrPC knockout mice and literature data in different cell types [24–26]. Similar results were obtained using an additional glioblastoma cell line (U251). As shown in Fig. 3j the positive HOP effect upon proliferation was also abrogated by HOP230–245 peptide in U251 PrPC-expressing cells, as well as being observed with U87 (Fig. 3g). Neurospheres from the PrPC knockout U251 cell line present a very low proliferation rate when compared to parental cells (Fig. 3j). Together these findings indicate that PrPC-HOP interaction is able to sustain the proliferation of GSCs of distinct glioblastoma cell lines.
HOP silencing impairs GSC proliferation
PrPC and HOP depletion decreases GSC tumorigenicity in vivo
Neurosphere cells expressing different levels of PrPC and/or HOP were injected subcutaneously into Balb/c nude mice flanks and tumor growth was evaluated. As depicted in Fig. 4e, cells expressing PrPC grew rapidly, while ablated cells for PrPC (PrP2) and/or HOP (HOPKD) showed slower or no tumor formation. Histological sections of resected tumors showed that PrPC or HOP silencing caused cell proliferation reduction, as depicted by Ki-67 nuclear immunostaining (Fig. 4f and g). These data support our in vitro assays and highlight the importance of PrPC and HOP in tumor maintenance through the modulation of GSC proliferation.
Self-renewal of GSCs depends on PrPC expression and its interaction with HOP
As previously described, PrPC ablation decreases neurosphere number (Fig. 5a) probably by downregulating essential molecules involved in stem cell maintenance (Fig. 2), supporting its previously suggested role in stemness. The smaller size of neurospheres formed by PrPC-depleted cells (compared to their counterparts) suggests a putative role for PrPC in GSC cell adhesion maintenance (Fig. 5b; Additional file 1: Figure S1A). To address this issue, the expression of E-cadherin and β-catenin, key components of adherens junctions, was evaluated in PrP2 neurospheres and parental control populations. The expression of E-cadherin is associated with PrPC on the cell surface; this was more obvious for the parental population, where there was higher expression of E-cadherin (Fig. 5c) and a clear double staining for both proteins in neurosphere cells (Fig. 5d). Although the cell membrane of parental cells had high E-cadherin expression, PrP2 had abundant cytoplasmic E-cadherin (Fig. 5e). Parental and PrP2 neurospheres expressed β-catenin abundantly at similar levels. However, there was an intense co-localization of β-catenin and PrPC in the same membrane domain of parental neurospheres, while in PrP2 neurospheres the β-catenin expression was diffuse (Fig. 5f). This may suggests that PrPC plays a role in E-cadherin recruitment to the cell membrane and, consequently, on β-catenin engagement.
Due to its putative function as a scaffold protein and multiprotein assembly platform on the cell surface, we also tested if additional anchorage-dependent cell processes, such as migration, was altered in PrPC-depleted cells. Additionally, migration on laminin is impaired in PrPC-silenced cells compared to parental neurospheres (Fig. 5g and h; Additional file 1: Figure S1B). Moreover, the surface expression of integrin α6β1, a laminin receptor, was verified. Integrin α6 expression is clearly more evident in parental neurospheres compared to PrP2 (Fig. 5i) and is co-expressed with PrPC, while integrin β1 expression is apparently similar in both populations (Fig. 5j). However, a high co-localization of integrin β1 and PrPC was observed (Fig. 5k), suggesting that PrPC plays a key function in enhancing cell surface stability of cell adhesion molecules, thereby modulating the invasive process.
In this study, the role of the PrPC-HOP complex in the regulation of GSC biology was evaluated. First, we demonstrated that our model of neurospheres expressed several markers of stemness. The expression of CD15, CD133, Oct4, Musashi-1, and Sox2 was increased in neurospheres compared to a non-stem condition (monolayer), validating our experimental model (Fig. 1). PrPC expression was higher in neurospheres than in monolayer cultures (Fig. 2a) and, since the enrichment of GBM cultures with stem cells leads to more malignant tumors in vivo , these data support previous results from our group which show that PrPC expression is correlated with tumor aggressiveness [6, 15].
PrPC expression seems to be associated with stem-like properties, since its silencing led to a differentiated expression of stem cell markers. Compared to parental neurospheres, CD133 expression decreased and Sox2 expression became undetectable in PrPC-depleted populations (Fig. 2e and f). Previous studies have shown that CD133 and Sox2 are exclusively expressed at perinecrotic and perivascular regions associated with stem-like cell pools, and that nestin and Musashi-1 are homogeneously expressed across the tumor, identifying precursors . PrP2 neurospheres expressed Musashi-1 in the cytoplasm in contrast to parental neurospheres, where it segregated preferentially to cell nuclei (Fig. 2f). Nuclear expression of Musashi-1 has been associated with activation of the Notch pathway in gliomas [29, 30] which, in turn, may lead to increased tumor malignancy via induction of proliferation and therapy resistance . The expression of nestin was similar in parental and PrP2 neurospheres (Fig. 2f). Expression of differentiation markers is more evident in PrP2 neurospheres compared to the parental population after serum stimuli (Fig. 2g). Together, our results suggest that parental neurospheres have a stem-like phenotype with some precursor cells, and that the PrP2 population has precursors and cells committed to a specific phenotype. Indeed, the function of PrPC in stem cell biology has been broadly studied. PrPC has been described as an important molecule for neural commitment and for the proliferation of precursors . In tumor stem-like cells, PrPC promotes proliferation and migration . PrPC interacts with the cell surface protein CD44, a marker for several types of cancer stem-like cells  that associates with tumor-initiating and metastatic capacities and promotes epithelial-mesenchymal transition (EMT) and tumor growth after resection .
A possible interaction between PrPC and CD133 was also observed given their co-expression and localization on the cell surface of parental neurospheres (Fig. 2b and c). PrPC and CD133 were previously shown to localize to the same membrane domains (lipid rafts), modulating differentiation and stemness, respectively . Additionally, a reduction in cell surface expression of CD133 associated with PrPC was observed after copper stimulus, suggesting PrPC as a carrier for CD133 internalization (Fig. 2c and d). PrPC is constitutively endocytosed via clatrin-coated pits  and copper ions reversibly stimulate this endocytic pathway . CD133 has been shown to affect the clathrin-endocytosis process  and trafficking down the endosomal and lysosomal pathway for degradation . Remarkably, the cytoplasmatic domain of CD133 binds β-catenin and the downregulation of CD133 increases β-catenin degradation and impairs tumor growth in vitro and in vivo . Indeed, the β-catenin localization was perturbed in PrPC-depleted cells (Fig. 5f), suggesting the central role of PrPC in the stabilization of the signaling module on the cell surface.
PrPC-depleted populations formed less secondary neurospheres (Fig. 5a), suggesting that withdrawing PrPC impairs self-renewal ability. This supports the hypothesis that PrPC can act as an important player in stemness maintenance and that its dowregulation induces cell line commitment, corroborating recent evidence that showed a less oncogenic phenotype in PrPC-depleted GSCs . Thus, it is possible that PrPC acts as an essential molecule for GSC biology and is capable of maintaining an undifferentiated state in this GBM subpopulation and, since its expression may indicate tumor enrichment with stem-like cells, it may be used as a tumor progression marker. Interesting, the effect of temozolomide, a common chemotherapeutic agent for brain tumors, is enhanced in PrPC-depleted glioma cells, supporting PrPC as an effective target for GBM .
As a number of studies show that PrPC acts as a scaffold protein, assembling signaling platforms on the plasma membrane to elicit several biological processes including in stem cells (reviewed in [8, 42]), we looked for alterations in the expression of cell adhesion proteins on the cell surface of GSCs. We observed a decrease in E-cadherin and integrin α6 expression on the cell surface of PrPC-silenced populations (Fig. 5c, d and i), and detected E-cadherin in the cytoplasm (Fig. 5e). The expression of these proteins was also associated with PrPC, as E-cadherin- or integrin α6-positive cells were almost exclusively positive for PrPC (Fig. 5d and i). Integrin β1 expression was detected in both parental and PrP2 neurospheres, and co-localized with PrPC on the cell surface (Fig. 5j and k). In addition, GSC migration on laminin was impaired in PrPC-depleted cells (Fig. 5g and h). Indeed, it has been demonstrated that PrPC participates in E-cadherin recruitment to the cell surface  with no significant differences verified in transcripts levels [43, 44]. Interestingly, it was demonstrated that PrPC-null mice present increased paracellular permeability, with lower levels of E-cadherin, desmoplakin, occluding, and other proteins related to cell-cell junctions in intestinal tissues . Furthermore, it was reported that PrPC is able to regulate β1 integrin adhesiveness modulating ligand-induced changes in integrin activation ; however, its depletion had no effect on total β1 integrin expression levels . These data suggest that PrPC may be capable of recruiting cell adhesion molecules to the cell surface of GSCs, raising the hypothesis of PrPC modulating invasion-related processes.
We also reported that GSCs have high expression of HOP and PrPC (Fig. 3b) and described the HOP-PrPC engagement on their plasma membrane (Fig. 3a). HOP expression and secretion was similar in parental, PrP1, and PrP2 populations (Fig. 3c–f), as demonstrated previously by Santos and collaborators in murine wild-type and PrPC-null neurospheres . We demonstrated that HOP is able to modulate GSC proliferation (Fig. 4d) and self-renewal, depending on its interaction with PrPC (Figs. 3g, 4d, and 5a); inhibiting the PrPC-HOP interaction with a HOP peptide, which mimics the PrPC binding site, abrogates the effects of recombinant HOP (Figs. 3g and 5a). Similar effects were also observed in another glioblastoma cell line (U251) confirming the importance of these complexes in GSC biology (Fig. 3j). Another HOP peptide (anti-TPR), which inhibits the HOP-Hsp90 interaction, has been described to induce cell death in several cancer cell lines  and produce a cytotoxic effect in glioblastomas , highlighting HOP as a potential target for GBM therapy. Previous reports  as well as data from this study (Fig. 3e and h) also described that PrPC and secreted HOP interact on the cell surface of human glioblastoma cell lines and modulate GBM progression by promoting proliferation through activation of the Erk1/2 pathway . Remarkably, a higher basal phosphorylation of Erk was found in PrPC-depleted cells when compared to their counterparts (Fig. 3h and i), confirming our prior data in retinae  and hippocampal neurons , as well as those from other authors in adult brain and cerebellum extracts . This feature suggests that high activity of Erk could be a compensatory effect for prion protein ablation . Indeed, the targeting of stress response proteins has remarkable potential for the development of molecular treatments. Simultaneous inhibition of expression of both HOP ligands Hsp70 and Hsp90 reduced proliferation and promoted apoptosis in GBM cell lines in vitro . Furthermore, HOP performs an important function in compensatory mechanisms of stress responses in tumor cells , further supporting its relevance in GBM maintenance.
Finally, we observed that PrPC and/or HOP knockdown affects the proliferative and tumorigenic capacity of GSCs (Fig. 4e and f) in vivo, and supports PrPC and HOP molecules as potential new targets for developing more efficient therapeutic strategies.
Major commitments for developing novel therapeutic strategies for GBM are under way, as this is an extremely aggressive type of cancer. In particular, several attempts to therapeutically eradicate GSCs have been made, as these cells have well-established characteristics in tumors. Our findings support the concept that PrPC, HOP, and their complex are important for GSC biology, regulating essential mechanisms for tumor maintenance. Therefore, they represent a novel target for developing new treatments for GBM or for improving the efficacy of current therapies by targeting GSCs.
Basic fibroblast growth factor
Bovine serum albumin
Epidermal growth factor
Glioblastoma stem-like cell
Hsp70/90 organizing protein
Neural stem cell
- PrPC :
Cellular prion protein
The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (11/13906-2, 2009/14027-2, 13/19860-0, and 15/04122-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (467566/2014-3), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for financial support. We are also very grateful to Carlos Nascimento for technical assistance, Dr. Andrew Hill (La Trobe University, Australia) for providing shRNA-PrP constructs used in this study, and Dr. Martín Roffé (A.C. Camargo Cancer Center) for excellent technical assistance with the CRISPR/Cas9 system.
Fundação de Amparo à pesquisa do Estado de São Paulo (2013/19860-0 to RPI; 2009/14027-2 to VRM; 2011/13906-2 to MHL; and 2015/04122-9 to MBP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (467566/2014-3 to VRM and TGS), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Institutional Scholarship to RPI).
Availability of data and materials
RNA design for CRISPR/Cas9 provided by Optimized CRISPR Design (http://crispr.mit.edu/) using PrPC gene sequence (NM_000311.3).
RPI: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; MBP: collection and/or assembly of data; LC; manuscript writing; VRM: manuscript writing, provision of study material, and financial support; TGS: conception and design, data analysis and interpretation, and manuscript writing MHL: conception and design, data analysis and interpretation, manuscript writing, financial support, provision of study material, and final approval of manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Institutional guidelines for animal welfare were followed and the study approved by the Animal Ethics Committee of the Institute of Biomedical Sciences/University of Sao Paulo (book 03, page 15, and protocol number 002 of 04/03/2014).
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