Disruption of Prion Protein&Ndash

ORIGINAL ARTICLE Disruption of prion protein–HOP engagement impairs glioblastoma growth and cognitive decline and improves overall survival

MH Lopes1,2,3,12, TG Santos1,3,12, BR Rodrigues1,3, N Queiroz-Hazarbassanov1,3, IW Cunha4, AP Wasilewska-Sampaio1,3, B Costa-Silva1,3, FA Marchi1,3,5, LF Bleggi-Torres6,7, PI Sanematsu8, SH Suzuki8, SM Oba-Shinjo9, SKN Marie9, E Toulmin10, AF Hill10 and VR Martins1,3,11

Glioblastomas (GBMs) are resistant to current therapy protocols and identiﬁcation of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)–Hsp90-organizing protein (HOP) with cellular prion protein (PrPC) triggers a large number of trophic effects in the nervous system. We found that both PrPC and HOP are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grades I–III. High levels of PrPC and HOP were associated with greater GBM proliferation and lower patient survival. HOP–PrPC binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase C and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrP binding site (HOP230–245) abrogates this effect. PrPC knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230–245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230–245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrPC–HOP engagement is a promising approach for GBM therapy.

Oncogene (2015) 34, 3305–3314; doi:10.1038/onc.2014.261; published online 25 August 2014

INTRODUCTION extracellular vesicles.11 Once in the extracellular milieu, HOP (at C Astrocytomas, the most common type of primary brain tumor, are amino acids (aa) 230–245) interacts with membrane-bound PrP 12,13 classified by the World Health Organization into four malignancy (at aa 113–128). C grades: I (pilocytic astrocytoma), II (diffuse astrocytoma), III PrP and HOP have been implicated in cancer progression. The C C (anaplastic astrocytoma) and IV [glioblastoma (GBM)] that is expression of an altered form of PrP , pro-PrP , that interacts with the most aggressive subtype of adult human brain tumors.1 filamin A is associated with poorer clinical outcome and survival of 14 C GBM usually presents in the sixth or seventh decade of life patients with pancreatic ductal adenocarcinoma. Pro-PrP is also and carries an average prognosis of o12 months.2 It exhibits expressed in melanoma cells, although its role in melanomagen- 15 C a relentless malignant progression characterized by esis is unclear. In colorectal tumors, PrP may regulate glycolytic 16 C uncontrolled cellular proliferation, diffuse infiltration, propensity rates by modulating GLUT1 levels. PrP expression is higher in for necrosis, robust neovascularization and resistance to metastatic than in non-metastatic gastric cancer where it is traditional and newer targeted therapeutic approaches.3 This thought to promote adhesion, migration and invasion.17 aggressive phenotype is the result of a variety of genetic and Two independent studies demonstrated elevated expression of 18,19 epigenetic alterations that lead to the deregulation of intracellular HOP, with Hsp70 or Hsp90, in colon tumors. High levels of 18,19 signaling pathways.4 these proteins have a negative impact on patient survival. We showed previously that the interaction of heat-shock Elevated HOP expression has been observed in pancreatic and – protein 70 (Hsp70)–Hsp90-organizing protein (HOP, aka stress- ovarian tumors and in hepatocellular carcinoma.20 22 Secretion of inducible phosphoprotein 1, STI1 or STIP1) with cellular prion HOP by GBM cells and other cancer cells is thought to influence – protein (PrPC) leads to the activation of signaling pathways that cell proliferation, invasion and angiogenesis.7,22 24 Indeed, HOP promote neuron/glial cell survival and differentiation.5,6 Some of has been shown to be secreted together with Hsp90 by breast these pathways also modulate proliferation of GBM cells in vitro.7 cancer, pancreatic tumor and fibrosarcoma cells, making Hsp90- HOP is a 543-residue (66-kDa) co-chaperone that binds Hsp70 and bound HOP available to form extracellular active complexes with Hsp90, modulating their folding activities.8–10 Despite its cyto- molecules such as matrix metalloproteinase-2, a collagenase plasmic localization, HOP is constitutively secreted mainly in related to tumor invasiveness.23–25 HOP secreted by ovarian

1International Research Center, AC Camargo Cancer Center, São Paulo, Brazil; 2Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; 3National Institute for Translational Neuroscience and National Institute of Oncogenomics (CNPq/MCT/FAPESP), São Paulo, Brazil; 4Department of Pathology, AC Camargo Cancer Center, São Paulo, Brazil; 5Inter-institutional Grad Program on Bioinformatics, Institute of Mathematics and Statistics, USP—São Paulo University, São Paulo, Brazil; 6Department of Pathology, Federal University of Paraná, Curitiba, Brazil; 7Pelé Pequeno Príncipe Research Institute, Curitiba, Brazil; 8Department of Neurosurgery, AC Camargo Cancer Center, São Paulo, Brazil; 9Department of Neurology, School of Medicine, University of São Paulo, Brazil; 10Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia and 11Ludwig Institute for Cancer Research, São Paulo, Brazil. Correspondence: Dr VR Martins, International Research Center, AC Camargo Cancer Center, Rua Tagua 440, São Paulo 01508-010, Brazil. E-mail: [email protected] 12These authors contributed equally to this work. Received 8 July 2013; revised 3 July 2014; accepted 8 July 2014; published online 25 August 2014 Prion protein–HOP targeting in glioblastoma MH Lopes et al 3306 cancer tissues can be detected in peripheral blood, making it a (Figure 2b), as previously demonstrated in normal brain.13 As biomarker that, when found in association with CA125, allows for shown in Figure 2c, immunoblots of conditioned medium (CM) early detection of these tumors.22 When secreted, HOP also from cultured xenograft tumors confirmed the presence of associates with activin A receptor, type II-like kinase-2, activating secreted HOP (66 kDa) in the absence of CDK4 (confirming the SMAD (Sma- and Mad-related proteins) signaling, and thereby absence of cell lysis). ELISA quantification showed that the 26 inducing proliferation in ovarian tumor cells. concentration of the secreted HOP increased with increasing We found that in human GBM cells in vitro, HOP binds C tumor size (Figure 2d). specifically to PrP and modulates proliferation through extra- The expression of PrPC in U87 cells was knocked down by cellular-signal-regulated kinase (ERK1/2) and phosphatidy- infection with lentiviral particles carrying anti-PrPC short hairpin 7 – C linositide 3-kinase (PI3K) pathways, suggesting that HOP PrP RNA (shRNA) sequence 1 (U87 shRNA 1) direct against mouse binding could be an important therapeutic target in GBMs. It is mRNA, used as a control, and sequence 2 (U87 shRNA 2) against noteworthy that we demonstrated that mice bearing orthotopic 27 human mRNA. Flow cytometry analysis (Figure 2e) showed that xenograft GBM exhibit early cognitive decline, consistent with shRNA 1-infected cells present a small inhibition (17.4 ± 2.7%) of the brain dysfunction and cognitive decline described for human PrPC levels owing to its partial homology with the human PrPC patients with brain tumors.28 sequence, whereas shRNA 1-infected cells show 84.6 ± 2.9% less Herein, the expression of HOP and PrPC was evaluated in human PrPC than non-infected cells. Treatment with recombinant HOP glioma specimens, grades I–IV, and correlated with tumor proliferation and patient survival. The role of PrPC–HOP engage- induced proliferation in shRNA 1-infected cells and non-infected ment and the impact of diminishing it on tumor growth, cognitive cells, but had no effect on the shRNA 2-infected cells. In addition, C Δ performance and survival were evaluated in vivo using orthotopic treatment with HOP lacking the PrP binding site (HOP ) had no xenograft mouse models. effect on proliferation (Figure 2f). Control U87, shRNA 1-infected and shRNA 2-infected cells were orthotopically xenografted into nude mice and osmotic pumps RESULTS (OPs) were implanted 9 days later to deliver saline, recombinant Overexpression of HOP and PrPC in GBMs HOP, or HOPΔ for 14 days. Tumors derived from shRNA 2 cells Evaluation of HOP gene expression with quantitative PCR (qPCR) were smaller than those derived from non-infected or shRNA experiments demonstrated that GBM (grade IV) samples had 1 cells. HOP treatment caused substantial increases in tumor size higher HOP mRNA levels than non-neoplastic tissues and grade II in mice bearing the non-infected and shRNA 1 cells, but tumors astrocytoma samples (Figure 1a). According to q–PCR findings, a derived from shRNA 2 cells were unaffected. HOPΔ treatment did meta-analysis using the database from The Cancer Genome Atlas not affect tumor growth in any of the groups (Figure 2g). Mice C (TCGA) for GBM samples also showed increased levels of mRNA of implanted with shRNA 2-infected cells (84.6% suppressed PrP ) both HOP (Agilent: 2.0-fold; Affymetrix: 1.5-fold) and PrPC (Agilent: survived significantly longer than mice implanted with shRNA 7.01-fold; Affymetrix: 6.96-fold) when compared with non- 1-infected cells (17.4% suppressed PrPC) or non-infected cells neoplastic tissues (Table 1). Moreover, the protein– (Figure 2h). These results are in agreement with findings in human protein interaction network reveals that HOP (k = 21) and PRNP GBMs (Figures 1f and g) in that with high levels of HOP in U87 cells (k = 88) present a high connectivity degree (Supplementary Figure 1). (as observed in Figures 2f and g), PrPC levels correlate directly with Complementary immunohistochemistry experiments per- proliferation and correlate inversely with survival, indicating that formed in a different set of astrocytoma specimens organized in U87 cells can recapitulate the properties observed here in human tissue microarrays demonstrated significantly higher expression of tumors. both HOP (Figures 1b and c) and PrPC (Figures 1d and e) in GBM samples versus non-neoplastic and grade I–III astrocytoma HOP – peptide abrogates GBM proliferation mediated by HOP samples. 230 245 GBM samples expressing high levels (i.e., above the median) of We hypothesized that the HOP230–245 peptide that mimics the C 12 – C both HOP and PrPC presented a higher proliferation ratio (indexed PrP binding site could be used to disrupt HOP PrP interaction by Ki-67 labeling) than those with high levels of HOP and in GBMs, and, consequently, control tumor growth (Figure 3a). low levels of PrPC (Figure 1f), indicating that the two proteins When U87 cells were pretreated with HOP peptides (HOP230–245 or may act in cooperation to promote proliferation. Furthermore, control peptide HOP61–76) and then incubated with recombinant Kaplan–Meier survival analysis indicated that patients with high HOP, we found that HOP230–245 treatment inhibited proliferation, C levels of HOP and PrP had shorter survival than those with high whereas the control peptide (HOP61–76) had no effect (Figure 3b). levels of HOP and low levels of PrPC (Figure 1g). No difference in Similar results were obtained with A172 cells (Figure 3e), which survival was found in patients with lower HOP levels (Figure 1g). were originally used to describe the role of HOP in GBM These findings indicate that HOP and PrPC are upregulated in GBM proliferation,7 and also with two additional human GBM cell lines, and that elevated expression of both proteins within tumors is U118 and U251 (Figures 3c and d, respectively). To address the correlated with increased tumor proliferation and decreased activation of intracellular signaling triggered by HOP, cells were patient survival. treated with recombinant HOP and/or peptides and then submitted to immunoblotting to evaluate ERK1/2 phosphoryla- 7 HOP mediates tumor growth in a PrPC-dependent manner tion. As described previously, HOP induced ERK1/2 phosphoryla- – Our previous results showed that HOP secreted by a non- tion in GBM cell lines (Figures 3i k) and HOP230–245 peptide tumorigenic glioma cell line (A172) induces proliferation in a abolished this effect, whereas control peptide did not alter HOP- PrPC-dependent manner.7 Here, the proliferative effect mediated induced ERK1/2 phosphorylation. Proliferation assays performed by secreted HOP through membrane-attached PrPC was evaluated in the presence of ERK1/2 and PI3K inhibitors (U0126 and 7 in a tumorigenic human glioblastoma cell line (U87). Flow LY29004, respectively), as previously described in A172 cells, cytometry analysis in U87 cells confirmed the high expression of demonstrated that both pathways are involved in HOP-mediated PrPC at the cell membrane (Figure 2a). Immunofluorescence proliferation (Figures 3f–h) in GBM cells. Taken together, these analysis of intracranial U87-xenografted tumors showed that PrPC data support the notion that proliferation mediated by HOP–PrPC and HOP were highly and uniformly expressed and colocalized, association via ERK1/2 and PI3K signaling pathways is impaired by indicating that both proteins may interact in tumors in vivo HOP230–245 peptide in GBM cells.

Figure 1. PrPC and HOP are overexpressed in the GBM, which is associated with augmented tumor proliferation and reduced patient survival. (a) qPCR for HOP expression in non-neoplastic brain tissues (NN) and astrocytoma grades I–IV (IV = GBM). *Po0.05; **Po0.005; Kruskal–Wallis test followed by Dunn’s multiple comparison test. (b and d) Left panels: representative images of an NN and GBM spot immunolabeled for HOP and PrPC, respectively. Right panels: representative high magniﬁcation images of HOP and PrPC expression (scale bar = 170 μm). (c and e) Quantiﬁcation of HOP and PrPC expression in NN and astrocytoma grades I–IV (IV, GBM). *Po0.05; Kruskal–Wallis test followed by Dunn’s multiple comparison test. (f) GBM samples with high expression (above the median) of HOP (n = 43) were divided into low (below the median, n = 21, blue bar) and high (above the median, n = 22, red bar) PrPC-level subgroups and evaluated for proliferation index by measuring the % of Ki-67 positive nuclei. *P = 0.0305; Student’s t-test. (g) Kaplan–Meier survival curve comparing patients bearing GBMs with high HOP and low PrPC expression (n = 21, blue line) versus those with high HOP and high PrPC expression (n = 20, red line). Patients with low HOP levels were also evaluated and divided into low PrPC (n = 20, green line) or high PrPC (n = 22, brown line). High HOP–high PrPC × high HOP–low PrPC, P = 0.0106 and log-rank test.

Table 1. HOP and PrPC overexpression in GBM samples from TCGA

Gene Region Connectivity degree (k) Lopes et al. GBM Affymetrix (413 samples) fold-change GBM Agilent (403 samples) fold-change

HOP 11q13 21 UP 1.53 2.00 PRNP 20p13 88 UP 6.96 7.01 Abbreviations: GBM, glioblastoma; HOP, Hsp70–Hsp90-organizing protein; PRNP, prion protein; UP, up-regulated.

HOP230–245 reduces tumor growth of human GBM xenografts and and e) or OPs (Figures 4d and e). At 14 days postinoculation (p.i.) increases survival. (summary time line in Figure 4a), the mean tumor volume in mice ﬁ To evaluate the capacity of HOP230–245 to inhibit tumor growth treated with HOP230–245 was reduced signi cantly and in a dose- in vivo, mice were inoculated with U87 cells and treated with dependent manner compared with control mice given saline. saline or peptides using a guide-screw system (GS) (Figures 4a–c Tumor size in HOP61–76-treated animals was similar to that in

Figure 2. HOP interaction with PrPC increases proliferation and tumor growth. (a) PrPC expression in U87 cells determined by flow cytometry using anti-PrPC antibodies (uns = unstained). (b) Frozen sections from U87-derived tumors grown intracerebrally in nude mice were immunolabeled with anti-PrPC (red) and anti-HOP (green) antibodies. The nuclei were counterstained with DAPI (blue). High in situ HOP–PrPC colocalization is observed (yellow, merge). Calibration bar = 25 μm. (c) Immunoblot analysis of HOP secretion in CM from U87-derived tumors of different sizes maintained in culture for 48 h; anti-CDK4 was used as cell lysis control. *Po0.01. (d) ELISA quantification of HOP in CM from U87-derived tumors of different sizes maintained in culture for 48 h. *Po0.01. (e) U87 cells infected or non-infected (non-inf) with lentiviral particles carrying shRNA sequences (shRNA 1 or shRNA 2) to knockdown PrPC expression. Cells were labeled with anti-PrPC antibodies and analyzed by flow cytometry. Representative histograms are shown. (f) U87 shRNA 1, U87 shRNA 2 and non-infected cells were treated or not (CTR) with recombinant HOP or HOPΔ (0.2 μM) for 24 h, and proliferation was accessed by BrdU incorporation. *Po0.01; ANOVA followed by Tukey’s post hoc test. (g) U87 shRNA 1 and U87 shRNA 2 and non-infected cells (0.5 × 106) were xenografted into the brains of nude mice. After 9 days, mice were implanted with subcutaneous OPs to infuse saline, HOP (25 ng/μl), or HOPΔ (deletion of PrPC binding site, 25 ng/μl) directly into their tumors for 14 days. After treatment, the mice were killed and the tumors were measured. *Po0.05; ***Po0.001; ANOVA followed by Tukey’s post hoc test. (h) Kaplan–Meier survival curve of mice implanted with 0.5 × 106 U87 shRNA 1, U87 shRNA 2 and non-infected cells. Log-rank Po0.03 shRNA 1 vs shRNA 2; Po0.03 non-infected vs shRNA 2 and P40.05 non-infected vs shRNA 1.

Figure 3. HOP-induced proliferation in U87 cells is abrogated by HOP230–245 peptide. (a) Scheme illustrating the hypothesis of how C competition of HOP binding to PrP with HOP230–245 peptide could affect cell proliferation. (b–h) U87 (green bars), U118 (yellow bars), U251 (blue bars) or A172 cells were cultured 48h in serum-free medium and treated with 10% FBS, HOP or with HOP peptides (HOP230–245 or control HOP61–76), or U0126, or LY29004 or DMSO 0.5% at the indicated concentrations for 24 h, and then cell proliferation was measured by BrdU incorporation. Values are expressed as means ± s.e. *Po0.05; **Po0.01; ***Po0.001; ANOVA, followed by Tukey’s post hoc test. (i–k) ERK1/2 activation in U87 (i), U118 (j) or U251 (k) cells treated with 10% FBS, HOP or with HOP peptides (HOP230–245 or control HOP61–76) for 15 min. The relative values of ERK1/2 activation were represented by the ratio of p-ERK and total ERK1/2. Blots are representative of six independent experiments. saline controls, demonstrating specificity of the HOP230–245 U251 tumors (Supplementary Figure 2). The results suggest that peptide on tumor growth inhibition (Figure 4a). HOP230–245 treatment decreases tumor size when compared with Histological sections of resected tumors showed that HOP230–245 HOP61–76 peptide treatment (Supplementary Figure 2c), similar to treatment caused a fivefold reduction in cell proliferation (Ki-67 what was described for U87 tumors. Taken together, these data labeling) and a 2.2-fold increase in apoptosis (cleaved-caspase-3 indicate that HOP230–245 peptide can decrease GBM growth in vivo labeling) compared with controls treated with HOP61–76 (Figures 4b and extend animal survival. and c). Intratumoral microvessel density (CD31 labeling) was not significantly affected by HOP230–245 treatment (Figures 4b and c). HOP230–245 preserves cognition in mice bearing orthotopic U87 A tumor growth curve pointed to day 9 p.i. as the appropriate xenografts day to initiate HOP230–245 treatment of pre-established tumors (Figure 4d), since that was when tumor volume could be The same groups of peptide-treated mice used in the above measured easily (volume range, 6.6–11.2 mm3) and animals did survival experiments (Figure 4e) were subjected to OR cognitive not present neurologic effects for the subsequent 2 weeks. In testing. At day 9 p.i. (pretreatment), mice in the GS groups, despite having substantial tumors (~8 mm3), were able to recognize new animals implanted with OP for intratumor delivery of HOP230–245 3 objects in a 90-min short-term memory (STM) test (memory or HOP61–76, mean tumor volume was 8.0 ± 3.4 mm at day 9 p.i. (pretreatment). Tumor growth was reduced in animals that evidenced by exploration time; Figure 5a). Mice treated with 3 HOP – continued to recognize new objects until day 25 p.i., received HOP230–245 (26.6 ± 5.0 mm ) for 14 days (day 23 p.i.) 230 245 relative to that observed in animals that received the control (Figure 5b), whereas HOP61–76-treated mice exhibited cognitive 3 fi peptide HOP61–76 (50.5 ± 8.7 mm for the same time (Figure 4d). de cits at day 12 p.i. (Figure 5c), almost 2 weeks before the onset U87 tumor-bearing mice treated with HOP230–245 (delivered of neurologic signs. A GS-implanted control mice (without tumor) via GS every 2 days or via an OP continuously, both starting from exhibited normal behavior (Figure 5d). day 9 p.i.) showed grade 4 neurologic signs (see Materials and Mice in the OP groups showed no evidence of cognitive deficits methods) later than HOP61–76-treated mice. Moreover, intratu- in 90-min STM and 24-h long-term memory (LTM) tests of object moral administration of HOP230–245 using either GS or OP recognition (OR) on days 8/9 p.i. (Figure 5e). Mice treated with increased animal survival compared with the HOP61–76 group HOP230–245 continued to show normal cognitive performance at (Figure 4e), suggesting that HOP230–245 peptide can decrease GBM days 13/14, 17/18 and 21/22 p.i. Conversely, mice treated with growth in vivo and extend animal survival. The effect of HOP230–245 HOP61–76 had impaired STM but normal LTM on days 13/14, and in reducing tumor growth was also evaluated in animals bearing then exhibited severe STM and LTM impairments on days 17/18

Figure 4. Intratumoral treatment of GBM xenografts with HOP230–245 peptide decreases tumor volume and modulates cell proliferation and 6 death. (a) U87 cells (0.5 × 10 ) were implanted into the right striatum of nude mice and intratumoral treatment with saline, HOP230–245 or HOP61–76 (0.3, 3 and 12 μg) was initiated on day 0 and followed every 2 days thereafter for 14 days using a GS system. Mice were killed and the tumors resected and measured. *Po0.05; ANOVA followed by Tukey’s post hoc test. (b) Representative images of tumor sections immunolabeled with markers for cell proliferation (Ki-67), apoptosis (cleaved-caspase-3) and vascularization (CD31). Nuclei were counterstained with DAPI (blue). Calibration bar = 100 μm. (c) Comparisons of labeling for Ki-67, cleaved-caspase-3 and CD31 between tumors treated with HOP230–245 or HOP61–76. The percentage of positive labeling area for Ki-67, cleaved-caspase-3 and CD31 among total DAPI-labeled cells was quantiﬁed. Values are expressed as means ± s.e. and are representative of at least three independent experiments. *Po0.01; Student’s t-test. (d) Mice with pre-established tumors (day 9 p.i.) received 12 μg of HOP61–76 or HOP230–245 peptides for 14 days using OPs. Animals were killed at day 23 p.i. for tumor volume evaluation. *Po0.05; **Po0.01; ANOVA, followed by Tukey’s post hoc test. (e) Kaplan–Meier survival curve of mice bearing U87 tumors. Treatment started at day 9 p.i. and was followed every 2 days with 12 μg HOP230–245 or HOP61–76 using a GS or OPs with a 28-day capacity. The treatment persisted until mice presented grade 4 neurologic signs, at which time they were killed. Differences between curves were evaluated by the log-rank test; P = 0.0006 for HOP230–245 vs HOP61–76 via GS and P = 0.0012 for HOP230–245 vs HOP61–76 via OP.

and 21/21 p.i. (Figure 5f). Thus, local treatment of tumors with grades I–III. Elevated levels of both HOP and PrPC were associated HOP230–245 peptide decreased the cognitive deficits that otherwise with augmented proliferation and reduced survival, suggesting a occurred with tumor growth. functional role of these proteins in tumor progression. These findings fit with previous reports of altered PrPC processing in pancreatic ductal adenocarcinoma and of elevated expression of DISCUSSION PrPC in colorectal tumors, melanoma and metastatic gastric cancer In this study, we showed that human GBMs present higher levels correlating with tumor relapse, poor clinical outcome, and of HOP and PrPC than non-tumor brain tissue or astrocytoma survival.14–17,29–31

Figure 5. Evaluation of cognitive deﬁcits in nude mice bearing U87 tumors treated with HOP230–245 peptide. OR was tested in mice treated with HOP230–245 or HOP61–76 peptide delivered by (a–d) a GS system every 2 days or (e and f) continuously by OP. At day 9 p.i. of U87 cells, mice (n = 14) were trained for 5 min with two identical objects (a). At the test, the animals were exposed to the familiar object (a) and novel ones (b and c) and exploration time for each object was measured 90 min (STM) or 24 h (LTM) later. (a and e) OR task at day 9 p.i. After test at day 9, animals were treated with (b) HOP230–245 or (c) HOP61–76. The test was repeated at days 12, 17 and 25 for animals that received HOP230–245 peptide and mice that received HOP61–76 peptide were tested at days 12 and 17 only, since most of them were dead by day 25. In each one of these days, the mice were trained again with new identical objects (c, e and g) and tested with novel objects (d, f and h) to assess for OR. (d) Animals with no tumors that were implanted with GS were also tested for OR. A second training phase was conducted with the same group of mice using a new set of objects (c and d). (f) Mice at day 9 p.i. of U87 cells received OP to deliver HOP230–245 or HOP61–76 and had STM evaluated on days 13, 17 and 21 p.i. and LTM tested on days 14, 18 and 22 p.i. The exploration time of familiar objects (e, h and k) was compared with that for novel ones (f, g, i, j, l and m). *Po0.0001; ANOVA followed by a Tukey's post hoc test.

Our findings also extend prior evidence showing that To isolate the role of the PrPC–HOP interaction, we conducted pancreatic,14 hepatocellular21 and ovarian22 carcinomas express in vitro and in vivo experiments using GBM cells with knocked high levels of HOP and that HOP depletion can reduce invasion down PrPC or mutant HOP (PrPC binding site deleted) and found 20,21,24 and proliferation of highly aggressive cell lines. Further- that the availability of the PrPC–HOP complex was associated with 32 more, it has also been shown that specific inhibitors or a tumor proliferation and reduced survival. Importantly, these 33 competitor peptide for the Hsp90-HOP interaction inhibit tumor experimental data are in agreement with our patient data. development, pointing to the Hsp90–HOP complex as a poten- We used the HOP230–245 peptide, which competes with full- tially selective anticancer therapeutic target.33,34 Mechanistically, C 12 35 11 length HOP for PrP binding, to test whether blockage of secreted HOP carried by exosome-like extracellular vesicles HOP–PrPC association could interfere with tumor growth. We may promote PrPC-dependent proliferation via activation of PI3K 7 found that HOP230–245 inhibited proliferation of four GBM cell lines and ERK1/2 or by binding to activin A receptor, type II-like kinase- in vitro. Moreover, intratumor delivery (a successfull approach 2 and activating the SMAD-ID signaling.26 used for other drugs in phase I and II clinical trials)3,40,41 of Remarkably, our results demonstrate that PrPC is expressed in HOP – peptide also inhibited tumor growth in vivo and U87 cells and that its knockdown decreases proliferation in 230 245 fi improved survival of the animals implanted with GBM cells. Thus, xenografted tumors and increases animal survival. These ndings fi are consistent with reports showing that PrPC inhibition decreases con rming the role of HOP230–245 peptide in the inhibition of GBM fi growth and induces programmed cell death in glioma36 and growth. It is important to note that these ndings will require colorectal tumor cells,16 and sensitizes tumor cells to cytotoxic further validation using primary GBM cultures, since many GBM 37,38 histopathologic features are not fully recapitulated in cell lines.42 drugs. It has been suggested that HOP knockdown may C reduce invasion of pancreatic cancer cells by decreasing expres- It is important to consider that PrP –HOP interaction itself has 43 sion of its downstream target gene matrix metalloproteinase-224 been implicated in self-renewal of neural stem cells. On the and by reducing pseudopodia formation and migration in breast other hand, temozolomide treatment commonly given to patients cancer.39 We did not test HOP knockdown in this study, since this with GBM induces PrPC overexpression44 and also selects a subset approach would not allow distinguishing effects on HOP of tumor stem cells,45 which might inadvertently contribute to co-chaperone activity from effects on the HOP–PrPC interaction. GBM recurrence. It remains to be determined whether the

© 2015 Macmillan Publishers Limited Oncogene (2015) 3305 – 3314 Prion protein–HOP targeting in glioblastoma MH Lopes et al 3312 PrPC–HOP complex also favors GBM stem cell self-renewal; if so, HSCOREs were as Σ(i × Pi), where Pi is % positive pixels and i is the staining fi – – HOP230–245 could potentially block this effect. intensity classi cation (0 3), yielding an HSCORE range of 0 300. The Finally, it is noteworthy that we found that HOP230–245 appeared percentage of positive Ki-67 nuclei was calculated according to the internal to prevent tumor-induced cognitive deficits in tumor-bearing algorithm of with ImageScope (IHC Nuclear v.1, Vista, CA, USA). mice. This beneficial effect was probably due to inhibition of tumor growth as demonstrated previously.27 This is particularly Proliferation and survival important since cognitive performance is increasingly valued as HSCOREs for PrPC and HOP expression in samples from 91 patients with secondary end point in clinical trials.46 On the other hand, a few GBM were compared with the % of Ki-67 positive nuclei. Samples were years ago, we demonstrated that HOP230–245 peptide had trophic divided into high- and low-expression groups, and split at the HSCORE properties13 and was able to improve memory formation when median. Associations between Ki-67 and PrPC and HOP expression C infused directly into the hippocampus.47 However, it is unlikely groupings were performed. PrP and HOP HSCOREs were used to that the present results can be attributed to peptide diffusion to construct Kaplan–Meier postsurgery survival curves. other brain regions such as the hippocampus. It is possible, although, that HOP230–245 peptide could protect neurons against Proteins and peptides apoptosis or degeneration caused by toxic antitumor drugs and Mouse recombinant HOP or HOPΔ (deletion mutant for amino acids radiotherapy.48 230–245) were purified12 and stripped of lipopolysaccharides with detoxi- Owing to intrinsic resistance of aggressive malignant astro- gel endotoxin removing gel (Thermo Scientific Pierce, Waltham, MA, USA). cytomas to established therapies, our ability to improve the Human HOP230–245 peptides (ELGNDAYKKKDFDTAL) and amino-terminal survival of these patients depends largely on strategies to target peptide HOP61–76 (GCKTVDLKPDWGKGYS) were synthesized by GenScript tumor cell resistance and the identification of novel antitumor (Piscataway, NJ, USA). agents. The advancement of new approaches to treat patients with GBM tumors may depend on identifying tumor-expressed Cultures molecules that are associated with tumor growth and developing Human U87MG, A172 and U118 cell lines were purchased from American new approaches to inhibit them. The present results show that the Type Collection (ATCC, Manassas, VA, USA). U215 cell were kindly provided PrPC–HOP complex is a key element in GBM proliferation. These by Drs Sueli Oba Shinjo and Suely Marie. Cells were cultured in Dulbecco's findings further suggest that PrPC–HOP complex formation may modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). Aseptically resected tumor specimens were be blocked by HOP230–245 peptide or peptide-mimetic drugs, and cultured for 48 h in Dulbecco's modified Eagle's medium F12 supplemen- thus point to a novel approach for treating patients with GBM, μ which with currently available treatments have a very poor ted with B27 and gentamicin (40 g/ml). The CM was centrifuged (2000 g) and filtered. prognosis. Cell proliferation assay MATERIALS AND METHODS Cells were cultured in Dulbecco's modified Eagle's medium plus 10% FBS qPCR at 37 °C for 12 h. Cells were washed and maintained in Dulbecco's modified Eagle's medium F12 for 48 h, and then treated for 24 h with 0.2 μM Fresh surgical astrocytoma and non-neoplastic brain tissue samples were Δ μ from University of São Paulo (approved by the Ethics committee—691/05). recombinant HOP and HOP , 0.2 or 8 M HOP or 10% FBS or RNA was extracted with RNeasy Mini Kits (Qiagen, Venlo, Netherlands) and pharmacologic inhibitors of ERK1/2 and PI3K (U0126 and LY29004, respectively; Calbiochem, San Diego, CA, USA). Cells were exposed to subjected to reverse transcription. Relative expression levels were μ fi determined by qPCR (ABI Prism 7500; Applied Biosystems, Foster City, bromodeoxyuridine (BrdU) (35 M) 2 h before xation and were processed fl CA, USA). A geometric mean of the expression of reference genes (BCRP, for immuno uorescence. The percentage of BrdU-positive nuclei was HPRT and GUSB) was used as a quantitative standard for relative expression calculated. Proliferation assays for U118 and U251 cells were evaluated analysis. Primer sequences are listed in Supplementary Table 1. The using BrdU Cell Proliferation Assay Kit (Cell Signaling, Danvers, MA, USA) 1.73 − ΔΔCt equation was applied to calculate relative HOP expression, according to the manufacturer's instructions. where ΔΔCt = ΔCt tumor − mean ΔCt non-neoplastic brain tissues and ΔCt = Ct HOP − Ct geometric mean of reference genes. RNA interference Deficient lentiviral particles were produced in HEK293FT cells using Meta-analyses the ViraPower Lentiviral Expressing System (Invitrogen, Carlsbad, CA, USA). As validation purposes, we used two types of publicly available data sets The pLenti constructs for the shRNA sequence were as follows: ′ from TCGA (The Cancer Genome Atlas): gene expression measurements shRNA 1 for a PrP mouse sequence: 5 -CACCAGAACAACTTCGTGCACGA ′ using Agilent G4502A-07 and Affymetrix U133. For transcript-level analysis, CTCGAAAAGTCGTGCACGAAGTTGTTC-3 ; and shRNA 2 for the human we use TCGA's normalization and gene summaries. It was considered the sequence 5′-CACCGCGTCAATATCACAATCAAGCCGAAGCTTGATTGTGATATT probe to gene mapping provided by TCGA in the Array Definition Files. The GACGC-3′. U87 cells were infected (multiplicity of infection = 25) with C protein–protein interaction network was evaluated using the NAViGaTOR lentiviral vectors carrying two constructs targeting PrP (shRNA 1 and 2). software (Toronto, ON, Canada).49 After infection, cells were selected with Zeocin (600 μg/ml) for 10 days.

Patients and tissue microarray Orthotopic glioma model Two tissue microarrays were prepared as in Kononen et al.50 from 185 Female or male 8–10-week-old Balb/C nude mice (nu/nu; Taconics, Hudson, formalin-fixed, paraffin-embedded astrocytomas (from A. C. Camargo NY, USA) were used. Institutional guidelines for animal welfare and Cancer Center, São Paulo, Brazil) and 14 non-neoplastic samples (from experimental conduct were followed and the study approved by Animal surgically remediable patients with intractable mesial temporal lobe Ethics Committee of A. C. Camargo Cancer Center (process 025/08). A epilepsy). Samples were prepared after approval from A. C. Camargo guide-screw (Plastic One, Roanoke, VA, USA) was used for intracerebral Cancer Center Research and Ethics Committee (Process 1613/11). The tumor cell engraftment and intratumoral injections.52 Three days after clinicopathologic characteristics of patients are available in the screw implantation, 0.5 × 106 U87 cells were inoculated through the screw Supplementary Information. into the right striatum. Drugs were injected through the screw over 6 min. For immunohistochemistry, tissue microarrays were incubated overnight For dose–response curves, treatment was initiated on the same day as with the primary antibody (Supplementary Table 2) at 4 °C, after antigen inoculation and continued every 2–3 days over 2 weeks. For tumor assays, retrieval. Reactions were developed as described previously.51 Virtual slides treatment was initiated at day 9 p.i. and continued every 2 days over were created with the ScanScope System (Aperio Technologies, Vista, CA, 14 days or until animals presented neurologic symptoms. For treatment USA). Positive pixels were quantitated and staining intensity was classified using OPs, U87 (0.5 × 106) or U251 (1.5 × 106) cells were implanted into the as 0 (negative), 1 (weakly positive), 2 (positive) or 3 (strong positive). right striatum using a Hamilton syringe, and at day 9 p.i. (for U87-

Oncogene (2015) 3305 – 3314 © 2015 Macmillan Publishers Limited Prion protein–HOP targeting in glioblastoma MH Lopes et al 3313 implanted animals) or at day 34 (for U251-implanted animals), mice were behavior of the mice toward familiar and novel objects was quantified. anesthetized and OPs (Alzet, Cupertino, CA, USA) were implanted After the first OR session at day 9, mice were implanted with OP to deliver subcutaneously and peptides were delivered to the tumors. Neurologic peptides HOP230–245 or HOP61–76 and further OR sessions were performed 53 symptoms were assessed by modified neurologic scores: grade 0, none; at days 13/14, 17/18 and 21/22 p.i. for STM (3 h) and LTM (24 h) no-tumor, grade 1, tail weakness/paralysis; grade 2, hind leg para/hemiparesis; grade GS-implanted mice served as controls. 3, hind leg para/hemiparalysis; grade 4, tetraplegia/moribund/death. Animals were euthanized when they presented grades 3 and 4. All mice (n = 169) were euthanized by CO2 saturation. Statistical analysis Mean values represent at least three independent data sets; error bars Tumor measurement represent standard errors of the mean. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for multiple The volume (mm3) of the tumors derived from U87 cells were calculated as comparisons. Unpaired Student’s t-test was used to compare Ki-67-, (length × width2)/2 as described.27 The tumors were fixed in 4% CD31- and cleaved-caspase3-immunostained groups. Nonparametric paraformaldehyde, sectioned (10 μm) and processed for immunofluores- fi ANOVA Kruskal–Wallis test followed by Dunn’s multiple comparison test cence. Animals bearing U251 tumors have their brains removed, xed, fi paraffin-embedded and hematoxylin and eosin stained. Tissues were were used to analyze immunochemistry and qPCR data. Signi cance was o – evaluated by a pathologist and tumor areas were used to estimate tumor accepted at P 0.05. Differences between Kaplan Meier survival curves extension in serial sectioning. were evaluated with the log-rank test.

Immunofluorescence CONFLICT OF INTEREST Fixed cells were treated with 2 N HCl for 30 min followed by borate buffer The authors declare no conflict of interest. for 10 min. Cells were blocked, permeabilized, incubated with anti-BrdU biotinylated antibody (Millipore, Billerica, MA, USA) overnight and labeled with Strepta-Alexa 488 (Molecular Probes, Carlsbad, CA, USA) and 4',6- diamidino-2-phenylindole (DAPI) for 1 h. Sections were blocked for 1 h and ACKNOWLEDGEMENTS incubated overnight with primary antibody (Supplementary Table 2). This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Secondary antibody (Supplementary Table 2) was applied for 1 h, followed (FAPESP 09/14027-2, 07/08410-2 and 04/12133-6) and Conselho Nacional de by DAPI staining. Slides were mounted with Fluorsave (Calbiochem). Desenvolvimento Científico e Tecnológico (CNPq). Fellowships from FAPESP Sections were viewed under an Olympus immunofluorescence microscope (to TGS) (2009/51653-9), NGQ (2009/51751-0), BC-S (2008/55381-0), BRR (2010- (Center Valley, PA, USA). 13654-0, 2012/19019-0), APW-S (2010/20796-6), and from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (to APW-S) are gratefully Immunoblotting acknowledged. We are thankful for Maria Del Mar Inda and Severino da Silva Ferreira CM was prepared from 48-h cultures of xenograft tumors, mixed in for technical assistance. Drs Maria Dirlei Begnami, Victor Piana de Andrade and Martín Laemmli buffer, resolved in 10% sodium dodecyl sulfate–polyacrylamide Roffe contributed with helpful discussions. We thank the AC Camargo Biobank for gel electrophoresis and immunoblotted13 with actin (loading), IgG providing the astrocytoma samples used in this study. (negative) and CDK4 (lysis) controls (Supplementary Table 2). Cells were lysed with Laemmli sample buffer and extracts were used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% gels). Proteins REFERENCES were transferred to nitrocellulose membranes (GE Healthcare, Little 1 Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A et al. Malignant Chalfont, UK) and immunoblotted to detect phosphorylated (p)-(T202/ astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 2007; 21: Y204)-ERK1/2 and total ERK1/2. Bands were imaged with UVITEC system 2683–2710. and quantified using ImageJ software (NIH, Bethesda, MD, USA). Levels of 2 Ohgaki H, Kleihues P. The definition of primary and secondary glioblastoma. Clin the p-ERK1/2 were relativized to total ERK1/2. Antibodies were purchased Cancer Res 2013; 19:764–772. from Cell Signaling. 3 Westphal M, Lamszus K. The neurobiology of gliomas: from cell biology to the development of therapeutic approaches. Nat Rev Neurosci 2011; 12:495–508. Flow cytometry 4 Jones TS, Holland EC. Standard of care therapy for malignant glioma and its effect on tumor and stromal cells. Oncogene 2012; 31:1995–2006. U87 cells were incubated with primary and secondary antibodies 5 Linden R, Martins VR, Prado MAM, Cammarota M, Izquierdo I, Brentani RR. (Supplementary Table 2) for 30 min each at 4 °C. FACS analyses were Physiology of the prion protein. Physiol Rev 2008; 88: 673–728. performed with FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). 6 Martins VR, Beraldo FH, Hajj GN, Lopes MH, Lee KS, Prado MAM et al. Prion protein: orchestrating neurotrophic activities. Curr Issues Mol Biol 2010; 12:63–86. ELISA 7 Erlich RB, Kahn SA, Lima FRS, Muras AG, Martins RAP, Linden R, et al. STI1 Binding plates (96-well) were coated overnight at 4 °C with CM from U87 promotes glioma proliferation through MAPK and PI3K pathways. Glia 2007; 55: cultures or tumors. Washed wells were then filled with phosphate-buffered 1690–1698. saline (with 5% milk) and incubated with primary (2 h) and secondary (1 h) 8 Maciejewski A, Prado MA, Choy W-Y. (1)H, (15)N and (13)C backbone resonance antibodies at 37 °C. The assay was developed in orthophenilenediamine assignments of the TPR1 and TPR2A domains of mouse STI1. Biomol NMR Assign 2013; 7:305–310. solution (0.33 mg/ml 0.5 M citrate buffer, pH 5.2, 0.4% H2O2) for 5 min at 9 Lee C-T, Graf C, Mayer FJ, Richter SM, Mayer MP. Dynamics of the regulation of room temperature. The reaction was stopped with 4 M H2SO4 and the absorbance (490 nm) measured using a microplate reader (Bio-Rad, Hsp90 by the co-chaperone Sti1. EMBO J 2012; 31: 1518–1528. Hercules, CA, USA). 10 Muller P, Ruckova E, Halada P, Coates PJ, Hrstka R, Lane DP et al. C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation Object recognition balances. Oncogene 2013; 32: 3101–3110. Animals inoculated with HOP230–245 or HOP61–76 and implanted with GS or 11 Hajj GNM, Arantes CP, Dias MVS, Roffé M, Costa-Silva B, Lopes MH et al. The OP were evaluated for OR.27 They were habituated to an open-field unconventional secretion of stress-inducible protein 1 by a heterogeneous polyvinyl chloride plastic arena (30 × 25 × 20 cm3) with 20 min per day free population of extracellular vesicles. Cell Mol Life Sci 2013; 70:3211–3227. exploration for 4 days. Then, they were placed in the arena with two 12 Zanata SM, Lopes MH, Mercadante AF, Hajj GNM, Chiarini LB, Nomizo R et al. identical objects on day 9, 12, 17 or 25 p.i. and left to explore for 5 min. Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers STM was tested in a 5-min trial 90 min later, wherein one of the identical neuroprotection. EMBO J 2002; 21: 3307–3316. objects was replaced by a novel one. A second group of animals were 13 Lopes MH, Hajj GNM, Muras AG, Mancini GL, Castro RMPS, Ribeiro KCB et al. inoculated with tumor cells and 9 days p.i. trained as described previously, Interaction of cellular prion and stress-inducible protein 1 promotes but STM was tested at 3 h and LTM was tested at 24 h. At the test, new neuritogenesis and neuroprotection by distinct signaling pathways. J Neurosci objects replaced one of the identical ones used in training. The exploratory 2005; 25: 11330–11339.

© 2015 Macmillan Publishers Limited Oncogene (2015) 3305 – 3314 Prion protein–HOP targeting in glioblastoma MH Lopes et al 3314 14 Li C, Yu S, Nakamura F, Yin S, Xu J, Petrolla AA et al. Binding of pro-prion 34 Horibe T, Torisawa A, Kohno M, Kawakami K. Molecular mechanism of cytotoxicity to filamin A disrupts cytoskeleton and correlates with poor prognosis in induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells. Mol pancreatic cancer. J Clin Invest 2009; 119: 2725–2736. Cancer 2012; 11:59. 15 Li C, Yu S, Nakamura F, Pentikainen OT, Singh N, Yin S et al. Pro-prion 35 Lima FRS, Arantes CP, Muras AG, Nomizo R, Brentani RR, Martins VR. Cellular prion binds filamin A, facilitating its interaction with integrin 1, and contributes to protein expression in astrocytes modulates neuronal survival and differentiation. J melanomagenesis. J Biol Chem 2010; 285: 30328–30339. Neurochem 2007; 103: 2164–2176. 16 Li Q-Q, Sun Y-P, Ruan C-P, Xu X-Y, Ge J-H, He J et al. Cellular prion protein 36 Barbieri G, Palumbo S, Gabrusiewicz K, Azzalin A, Marchesi N, Spedito A et al. promotes glucose uptake through the Fyn-HIF-2α-Glut1 pathway to support Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonu- colorectal cancer cell survival. Cancer Sci 2011; 102:400–406. cleotides induces autophagy-dependent cell death in glioma cells. Autophagy 17 Pan Y. Cellular prion protein promotes invasion and metastasis of gastric cancer. 2011; 7: 840–853. FASEB J 2006; 20: 1886–1888. 37 Yu G, Jiang L, Xu Y, Guo H, Liu H, Zhang Y et al. Silencing prion protein in MDA- 18 Kubota H, Yamamoto S, Itoh E, Abe Y, Nakamura A, Izumi Y et al. Increased MB-435 breast cancer cells leads to pleiotropic cellular responses to cytotoxic expression of co-chaperone HOP with HSP90 and HSC70 and complex formation stimuli. PLoS One 2012; 7: e48146. in human colonic carcinoma. Cell Stress Chaperones 2010; 15: 1003–1011. 38 Meslin F, Conforti R, Mazouni C, Morel N, Tomasic G, Drusch F et al. Efficacy of 19 Ruckova E, Muller P, Nenutil R, Vojtesek B. Alterations of the Hsp70/Hsp90 adjuvant chemotherapy according to prion protein expression in patients with 18 – chaperone and the HOP/CHIP co-chaperone system in cancer. Cell Mol Biol Lett estrogen receptor-negative breast cancer. Ann Oncol 2007; :1793 1798. 2012; 17: 446–458. 39 Willmer T, Contu L, Blatch GL, Edkins AL. Knockdown of Hop downregulates RhoC 20 Walsh N, O’Donovan N, Kennedy S, Henry M, Meleady P, Clynes M et al. expression, and decreases pseudopodia formation and migration in cancer 328 – Identification of pancreatic cancer invasion-related proteins by proteomic cell lines. Cancer Lett 2013; : 252 260. analysis. Proteome Sci 2009; 7:3. 40 Allhenn D, Boushehri MAS, Lamprecht A. Drug delivery strategies for the treat- 436 – 21 Sun W, Xing B, Sun Y, Du X, Lu M, Hao C et al. Proteome analysis of hepatocellular ment of malignant gliomas. Int J Pharm 2012; :299 310. carcinoma by two-dimensional difference gel electrophoresis: novel 41 Quant EC, Wen PY. Novel medical therapeutics in glioblastomas, including protein markers in hepatocellular carcinoma tissues. Mol Cell Proteom 2007; 6: targeted molecular therapies, current and future clinical trials. Neuroimaging Clin 20 – 1798–1808. NAm2010; :425 448. fi 22 Wang T-H, Chao AA-S, Tsai C-L, Chang C-L, Chen S-HLee Y-S et al. Stress-induced 42 Joo KM, Kim J, Jin J, Kim M, Seol HJ, Muradov J et al. Patient-speci c orthotopic phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ. Cell Rep 2013; 3: 260–273. cancer cell proliferation. Mol Cell Proteom 2010; 9: 1873–1884. 43 Santos TG, Silva IR, Costa-Silva B, Lepique AP, Martins VR, Lopes MH. Enhanced 23 Sims JD, McCready J, Jay DG. Extracellular heat shock protein (Hsp)70 and Hsp90α neural progenitor/stem cells self-renewal via the interaction of stress-inducible assist in matrix metalloproteinase-2 activation and breast cancer cell migration protein 1 with the prion protein. Stem Cells 2011; 29: 1126–1136. and invasion. PLoS One 2011; 6: e18848. 44 Zhuang D, Liu Y, Mao Y, Gao L, Zhang H, Luan S et al. TMZ-induced PrPc/par-4 24 Walsh N, Larkin A, Swan N, Conlon K, Dowling P, McDermott R et al. RNAi interaction promotes the survival of human glioma cells. Int J Cancer 2012; 130: knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via 309–318. MMP-2 down regulation. Cancer Lett 2011; 306:180–189. 45 Chen J, Li Y, Yu T-S, McKay RM, Burns DK, Kernie SG et al. A restricted cell 25 Eustace BK, Jay DG. Extracellular roles for the molecular chaperone, hsp90. Cell population propagates glioblastoma growth after chemotherapy. Nature 2012; Cycle. 2004; 3: 1098–1100. 488:522–526. 26 Tsai C-L, Tsai C-N, Lin C-Y, Chen H-W, Lee Y-S, Chao A et al. Secreted stress- 46 Butowski N, Chang SM. Endpoints for clinical trials and revised assessment in induced phosphoprotein 1 activates the ALK2-SMAD signaling pathways and neuro-oncology. Curr Opin Neurol 2012; 25:780–785. promotes cell proliferation of ovarian cancer cells. Cell Rep 2012; 2:283–293. 47 Coitinho AS, Lopes MH, Hajj GN, Rossato JI, Freitas AR, Castro CC et al. Short-term 27 Wasilewska-Sampaio AP, Santos TG, Lopes MH, Cammarota M, Martins VR. The memory formation and long-term memory consolidation are enhanced by cel- growth of glioblastoma orthotopic xenografts in nude mice is directly correlated lular prion association to stress-inducible protein 1. Neurobiol Dis 2007; 26: with impaired object recognition memory. Physiol Behav 2013; 123C:55–61. 282–290. 28 Caine C, Mehta MP, Laack NN, Gondi V. Cognitive function testing in adult brain 48 Gehring K, Sitskoorn MM, Aaronson NK, Taphoorn MJB. Interventions for cognitive tumor trials: lessons from a comprehensive review. Expert Rev Anticancer Ther deficits in adults with brain tumours. Lancet Neurol 2008; 7: 548–560. 12 – 2012; : 655 667. 49 Brown KR, Otasek D, Ali M, McGuffin MJ, Xie W, Devani B et al. NAViGaTOR: 29 Sy M-S, Altekruse SF, Li C, Lynch CF, Goodman MT, Hernandez BY et al. Association Network Analysis, Visualization and Graphing Toronto. Bioinformatics 2009; 25: of prion protein expression with pancreatic adenocarcinoma survival in the SEER 3327–3329. 10 – residual tissue repository. Cancer Biomark 2012; :251 258. 50 Kononen J, Bubendorf L, Kallioniemi A, Bärlund M, Schraml P, Leighton S et al. 30 Antonacopoulou AG, Palli M, Marousi S, Dimitrakopoulos F-I, Kyriakopoulou U, Tissue microarrays for high-throughput molecular profiling of tumor specimens. Tsamandas AC et al. Prion protein expression and the M129V polymorphism of Nat Med 1998; 4:844–847. 49 the PRNP gene in patients with colorectal cancer. Mol Carcinogen 2010; : 51 Alvarenga AW, Coutinho-Camillo CM, Rodrigues BR, Rocha RM, Torres LFB, – 693 699. Martins VR et al. A comparison between manual and automated evaluations of 31 Antonacopoulou AG, Grivas PD, Skarlas L, Kalofonos M, Scopa CD, Kalofonos HP. tissue microarray patterns of protein expression. J Histochem Cytochem 2013; 61: POLR2F, ATP6V0A1 and PRNP expression in colorectal cancer: new molecules with 272–282. prognostic significance? Anticancer Res 2008; 28: 1221–1227. 52 Lal S, Lacroix M, Tofilon P, Fuller GN, Sawaya R, Lang FF. An implantable guide- 32 Pimienta G, Herbert KM, Regan L. A compound that inhibits the HOP–Hsp90 screw system for brain tumor studies in small animals. J Neurosurg 2000; 92: complex formation and has unique killing effects in breast cancer cell lines. Mol 326–333. Pharm 2011; 8: 2252–2261. 53 Weissert R, Wallström E, Storch MK, Stefferl A, Lorentzen J, Lassmann H et al. MHC 33 Horibe T, Kohno M, Haramoto M, Ohara K, Kawakami K. Designed hybrid TPR haplotype-dependent regulation of MOG-induced EAE in rats. J Clin Invest 1998; peptide targeting Hsp90 as a novel anticancer agent. J Transl Med 2011; 9:8. 102:1265–1273.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)