Expression of heat shock proteins in medulloblastoma

J Neurosurg Pediatrics 12:452–457, 2013

452 J Neurosurg: Pediatrics / Volume 12 / November 2013

©AANS, 2013

MedulloblastoMa (MB) is the most common malignant brain tumor in children, comprising nearly 20% of primary CNS tumors, and results

in high mortality rates and severe treatment-associated long-term side effects.1 Thus, novel therapeutic options for patients with these tumors are urgently warranted.

Levels of heat shock proteins (HSPs) have been found to be elevated in many human cancers and are impli-cated in tumor cell proliferation, differentiation, inva-sion, metastasis, death, and recognition by the immune system.4 They are potential targets for therapy since they can be targeted by drugs, or they may provide a pos-sible target for immunotherapy.4,5,25 Regarding brain tu-

Expression of heat shock proteins in medulloblastoma

Laboratory investigation

GeorGe A. Alexiou, M.D., Ph.D.,1 GeorGe VArtholoMAtos, Ph.D.,2 KAllioPi stefAnAKi, M.D., Ph.D.,3 AMAliA PAtereli, M.D., Ph.D.,3 lefKotheA DoVA, Ph.D.,2 AchilleAs KArAMoutsios, Ph.D.,2 GeorGe lAllAs, Ph.D.,4 GeorGe sfAKiAnos, M.D., Ph.D.,1 MAriA MoschoVi, M.D., Ph.D.,5 AnD neofytos ProDroMou, M.D., Ph.D.1

1Department of Neurosurgery, Children’s Hospital “Agia Sofia,” Athens; 2Hematology Laboratory–Unit of Molecular Biology, University Hospital of Ioannina, Ioannina; 3Department of Pathology, Children’s Hospital “Agia Sofia,” Athens; 4Flowcytogen Laboratories Ltd, Koropi, Athens; and 5Hematology Oncology Unit of 1st Department of Pediatrics, Athens University, Athens, Greece

Object. Medulloblastoma (MB) is the most common malignant brain tumor in children. Heat shock proteins (HSPs) comprise a superfamily of proteins that serve as molecular chaperones and are overexpressed in a wide range of human cancers. The purpose of the present study was to investigate the expression of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt by multiplex bead array assay of MBs. The results of HSP and Akt expression were correlated with MB subtype; immunohistochemical expression of Ki-67 index, bcl-2, and p53; and patients’ prognosis.

Methods. The authors retrospectively evaluated 25 children with MB who underwent surgery. Immunohisto-chemical analysis of Ki-67, p53, and bcl-2 expression was performed in all cases. By using multiplex bead array assay, a simultaneous detection of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt was performed.

Results. Medulloblastoma with extensive nodularity had significantly lower HSP27 (pSer15) expression (p = 0.039) but significantly higher HSP60 expression (p = 0.021) than classic MB. Large-cell MB had significantly higher HSP70 expression (p = 0.028) than classic MB. No significant difference was found between HSP27 (pSer82), HSP40, HSP90-α, Akt, or phospho-Akt expression and MB subtype. Large-cell MBs had significantly higher Ki-67 index compared with classic MBs (p = 0.033). When analyzing all MBs, there was a significant negative correlation between HSP27 (pSer15) and Ki-67 index (r = -0.475, p = 0.016); a significant positive correlation between HSP70 expression and Ki-67 index (r = 0.407, p = 0.043); and a significant positive correlation between HSP70 expression and bcl-2 index (r = 0.491, p = 0.023). Patients with large-cell MB had a worse survival than those with classic MB, but the difference did not reach statistical significance (p = 0.076).

Conclusions. A substantial expression of several HSPs in MB was observed. Given that HSPs represent an at-tractive strategy for anticancer therapy, further studies, involving larger series of patients, are obviously necessary to clarify the relationship of HSPs with tumor aggressiveness and prognosis.(http://thejns.org/doi/abs/10.3171/2013.7.PEDS1376)

Key WorDs • heat shock protein • Ki-67 • bcl-2 • p53 • medulloblastoma • children • immunohistochemistry • oncology • multiplex bead array assay

Abbreviations used in this paper: HSP = heat shock protein; MB = medulloblastoma; SHH = sonic hedgehog; WNT = wingless.

This article contains some figures that are displayed in color on line but in black-and-white in the print edition.

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Heat shock proteins in medulloblastoma

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mors, increased HSP expression has been reported.11,15 In pediatric brain tumors, HSPs have been studied by immunohistochemistry in MB, and high expression of HSP27, HSP70, and HSP90 has been found.13 The pur-pose of the present study was to investigate the expres-sion of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt in MB by using multiplex bead array assay. The results of HSP and Akt expression were correlated with MB subtype; immuno-histochemical expression of Ki-67, bcl-2, p53 index; and patient’s prognosis.

MethodsWe retrospectively evaluated data obtained in 25

children in whom MBs were treated surgically at Chil-dren’s Hospital “Agia Sofia” between January 2007 and January 2012. The study was approved by the institu-tional review board. All tumors were classified into the histological MB subtypes: classic; desmoplastic/nodular; extensive nodularity; and large-cell/anaplastic MB—ac-cording to the 2007 WHO classification. All patients who were more than 3 years of age received craniospinal ir-radiation and chemotherapy (vincristine, carboplatin, and etoposide alternating with vincristine, cyclophosphamide, and etoposide every 21 days). Children under 3 years of age received only chemotherapy (vincristine and carbo-platin alternating with vincristine and cyclophosphamide alternating with vincristine and methotrexate alternating with vincristine and cisplatin every 2 weeks). All patients were followed clinically during and after treatment.

Immunohistochemical AnalysisFour-micrometer-thick, formalin-fixed, paraffin-em-

bedded tissue sections were immunostained using the strep tavidin-biotin–horseradish peroxidase method (Su-persensitive Multilink Kit QD0005 L, Biogenex). Deparaf-finized sections were rehydrated through graded series of alcohol and then microwaved in 0.1 mol/L sodium citrate buffer solution (pH 6.0) for 3 × 5 minutes at 450 W to un-mask antigen epitopes. After treatment with 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase, the sections were incubated with the primary antibodies (Ki-67/MIB-1 [clone MIB-1, dilution 1/50, DAKO], p53 [clone DO-7, dilution 1/50, DAKO], bcl-2 [clone 100/D5, dilution 1/50, Novocastra/Leica], Synaptophysin [clone SY38, Monosan], neurofilaments [clone 2F11(70+200 kDa), Monosan], Neu-N [clone A60, Chemicon, Millipore], and b-tubulin III [clone TU-20, AbD serotec]) for 50 minutes at room temperature. Afterward, the sections were incubated with ready-to-use biotin-labeled secondary antibody and streptavidin peroxidase for 20 minutes each. Tissues were then stained with 0.05% 3,3-diaminobenzidine tetrahydro-chloride diluted in DAB (3’3-diaminobenzidine) substrate and then counterstained with hematoxylin, dehydrated, and mounted. Tris buffer solution (pH 7.6) was used for rinsing the sections between incubation steps and the dilutions of primary antibodies. The immunohistochemical expression of Ki-67, p53, and bcl-2 was evaluated by 2 independent experienced pathologists (K.S. and A.P.). Based on pre-vious studies, the Ki-67 expression was considered high

when greater than 50%, and p53 and bcl-2 expression was considered high when greater than 15% and greater than 30%, respectively.22–24 Only Ki-67 and p53 nuclear stains were considered positive. Results were expressed as the percentage of positive tumor cells out of the total number of counted tumor cells (approximately 3000 counted cells) in the most stained areas. All cells with staining (whatever the intensity) were considered positive. Any discrepancy between the 2 pathologists was resolved by consensus.

Sample PreparationWe used frozen tissues from brain tumor specimens

that were verified by cryosections. For the tissue extracts, we followed the kit insert suggested protocol. In brief, we used an approximately 0.5-cm3 piece of tissue. We calcu-lated the cell concentration and lysed 1 million cells per assay as following: 1 ml of HSP/Chaperone Lysis Buffer supplement with protease inhibitor cocktail (Catalog no. P8340, Sigma) at a concentration of 0.5mL/mL, phospha-tase inhibitor cocktail (Catalog no. P2850, Sigma) at a con-centration of 10 ml/ml, and PMSF (phenylmethylsulfonate) (Catalog no. P7626, Sigma) at a concentration of 10mL/ml. The cells were homogenized with a pestle and passed through a 21-gauge syringe 5 times to ensure complete cell lysis. The extracts were transferred to a polypropylene tube vortex and incubated on ice for 30 minutes. Then, they were centrifuged at 16,000 × g for 20 minutes in a 4°C refrigerated microfuge. The supernatant was transferred to a labeled polypropylene tube. The collected supernatant is the tissue extract, which is ready for analysis. The resulting pellet was discarded. Alternatively, the tissue extracts can be frozen at -70°C and assayed at a later date. The same procedure was followed for peripheral blood mononuclear cells of blood samples, which were used as normal control in our analysis.

Multibead Analysis ProcedureWe quantified 8 different molecules (total Akt, Akt

phospho-Ser473, HSP60, HSP70, HSP90-α, HSP40, HSP27 phospho-Ser82, and HSP27 phospho-Ser15) per sample following the suggested procedure of Multibead HSP/Chaperone 8-plex kit (Catalog no. ADI-980-002, Enzo Life Sciences). Briefly for each sample we used 50 ml of cell extracts diluted with assay buffer (1:4). Each of the analyte-specific antibodies is captured in beads of the same size (5.4 mm) and different intensity in red fluo-rescence channel (FL4). The analysis of the results was performed with dedicated analysis software (MultiBead Analysis Software, Enzo Life Sciences). The sensitivity of each analyte, based on the lower-used standard, is as follows: Akt (0.2 ng/ml), HSP27 phospho-Ser82 (5.2 U/ml), HSP27 phospho-Ser15 (3.4 U/ml), HSP40 (0.2 ng/ml), HSP60 (3.4 ng/ml), HSP70 (0.2 ng/ml), HSP90-α (0.3 ng/ml), and Akt phospho-Ser473 (2.0 U/ml).8 For the analy-sis we used FACSCalibur Flow Cytometer equipped with 2 lasers (488 and 635 nm) and 6 parameters (Fsc, Ssc, and FL1–FL4).

Statistical AnalysisCorrelations among HSP27 phospho-Ser82, HSP27

G. A. Alexiou et al.


phospho-Ser15, HSP40, HSP60, HSP70, HSP90-α, Akt, phospho-Akt, and Ki-67/MIB-1, bcl-2, p53 indices of tumor tissue were analyzed statistically using Spearman analysis. Differences in patients’ ages, HSPs, Akt, phospho-Akt, Ki-67, bcl-2 and p53 expression between MB subtypes were estimated using the 2-tailed paired t-test. Overall survival was defined as the time from surgery to death or as the time to the last follow-up of the surviving patients. Survival curves were calculated and compared using the Kaplan-Meier method and the log-rank test. A 2-sided p value < 0.05 was considered statistically significant.

ResultsTwenty-five patients (17 males, 8 females, mean

age 6.86, range 1–14 years) had an MB. Histologically, 17 MBs were classic, 5 were large cell, 2 had extensive nodularity, and 1 was desmoplastic/nodular. Leptomen-ingeal and choroid plexus infiltration was observed in 19 (76%) and 15 (60%) cases, respectively. No significant difference was found between MB subtype and patient age. Table 1 summarizes the immunohistochemical ex-pression of Ki-67, bcl-2, and p53 according to MB sub-type. The Ki-67 index ranged from 30% to 70% (mean 48%). Large-cell MBs had a significantly higher Ki-67 index than classic MBs (p = 0.033). Expression of p53 was detected in 23 patients (92%) and ranged from 0% to 60% (mean 25.6%). No correlation was found between p53 expression and MB subtype. Expression of bcl-2 was assessed in 21 patients. It ranged from 0% to 80% (mean 42.4%) (Fig. 1). There was a trend toward higher bcl-2 expression in large-cell MB compared with classic MB (p = 0.054).

Table 2 summarizes the expression of all studied HSPs in MB according to histological subtype. No sig-nificant difference was found between HSP27 (pSer82), HSP40, HSP90-α, Akt, and phospho-Akt expression and MB subtype. Medulloblastomas with extensive nodular-ity had significantly lower HSP27 (pSer15) expression (p = 0.039) but significantly higher HSP60 expression (p = 0.021) compared with classic MBs. Large-cell MBs had significantly higher HSP70 expression (p = 0.028) than classic MBs. There was a trend toward higher HSP27 (pSer15) expression (p = 0.052) but lower HSP60 expres-sion (p = 0.053) in large-cell MBs compared to tumors with extensive nodularity.

When analyzing all MBs, a significant negative cor-relation between HSP27 (pSer15) and Ki-67 index (r = -0.475, p = 0.016) was observed. There was a significant positive correlation between HSP70 expression and Ki-67

index (r = 0.407, p = 0.043). A significant positive cor-relation between HSP70 expression and bcl-2 index (r = 0.491, p = 0.023) was also observed. Finally, there was a trend toward positive correlation between HSP40 and p53 expression (p = 0.062). After a mean follow-up period of 26.3 months (range 7 months to 63 months), 18 pa-tients were alive. The overall survival was 72%. Patients with large-cell MBs had a worse survival than those with classic MBs, but the difference did not reach statistical significance (p = 0.076). No other variable reached sta-tistical significance, probably due to the limited number of patients.

DiscussionThe present study demonstrated a substantial expres-

sion of HSP in MBs. Large-cell MBs had higher expres-sion of Ki-67, bcl-2, and HSP70 than classic MBs and were associated with worse survival. When analyzing all MBs, we observed a significant negative correlation between HSP27 (pSer15) and Ki-67 index. A significant positive correlation between HSP70 expression and Ki-67 index was demonstrated. Antiapoptotic HSP70 correlated significantly with bcl-2 index.

Medulloblastomas are the most common malignant pediatric brain tumor.1 Several histological variants have been described. Classic and nodular/desmoplastic MBs are the 2 major histological subtypes. The extensive nodular subtype and large-cell/anaplastic MBs are the least fre-quent.21,31 The latter has been associated with poor prog-nosis, similar to the results of the present study.10 Recent molecular analyses identified 4 distinct molecular subtypes within MBs depending on the predominantly activated sig-naling pathways.26,30 The 4 recognized subtypes were the sonic hedgehog (SHH) subgroup, the WNT subgroup, and 2 subgroups that arise from an undefined class of cerebellar progenitors. These 4 subgroups have distinct demographic features, clinical presentation, transcriptional profiles, ge-netic abnormalities, and clinical outcome and may permit a more targeted therapeutic approach.26 Nevertheless, the role of histopathology will certainly not be abolished and considered an important prognostic factor.19,32 Ellison et al. reported that all studied desmoplastic/nodular medul-loblastomas were SHH tumors, while the majority of WNT tumors had a classic phenotype.7 Large-cell/anaplastic tu-mors can be found in all 4 subgroups but are more common in Subgroup 3 of the molecular classification.32 C-myc and n-myc amplifications have been observed in 4%–17% of MBs and are correlated with the large-cell/anaplastic type and adverse prognosis.34

TABLE 1: Immunohistochemical expression of Ki-67, bcl-2 and p53 according to MB subtype

ExpressionMB Subtypes

Classic (n = 17)* Large-Cell (n = 5)* Extensive Nodularity (n = 2)* Desmoplastic/Nodular (n = 1)

Ki-67 35.3 ± 9.6 57.4 ± 19.1 42.5 ± 10.6 50bcl-2 29.3 ± 24 77.5 ± 5 49.4 ± 45.5 80p53 21.2 ± 13.1 32 ± 25.6 37.5 ± 31.8 40

* Values are presented as the mean ± SD.



455

Expression of HSP is induced by stress such as radia-tion, heat, anticancer agents, and oxidative stress. Mamma-lian HSPs has been classified according to their size into 6 families: HSP100, HSP90, HSP70, HSP60, HSP40, and small HSPs (15–30 kD) including HSP27.16 To the best of our knowledge, this is the first study in which such a large number of HSPs has been investigated in MBs. A previous study conducted by Hauser et al.13 investigated the expres-sion of HSP27, HSP70, and HSP90 in MBs of 65 patients by immunohistochemistry. The authors found that the ex-tent of expression of any of the HSP types was not signifi-cantly associated with known prognostic factors, histologi-cal subtype, or overall survival. However, HSP expression was only analyzed immunohistochemically. In our study we used multiplex bead array assay, which allows the quan-tification of multiple proteins simultaneously, in contrast to ELISA or Western blot analysis, which require a similar amount of sample, but only one protein can be analyzed.8 This technique is easily performed and reproducible. Fur-thermore, it is cost effective and time effective and mini-mizes the sample volume requirements.8

By inhibiting apoptosis, HSP27 protects against cell death triggered by various stimuli. It can be phosphor-ylated at 3 serine residues identified as Ser15, Ser78, and Ser82, with the last being the major site. Phosphorylation of the 3 serine residues of HSP27 has been shown to mod-ulate HSP27 functions.9 The Ser82 phosphorylation regu-lates oligomerization of the HSP27 and thus its biochemi-cal functions. The present study investigated, for the first

time, the site-specific phosphorylation of HSP27 at Ser82 and Ser15 in MBs. A significant negative correlation be-tween HSP27 (pSer15) and Ki-67 index was found. Fur-thermore, the antiapoptotic HSP70 was also investigated, and a significant positive correlation between HSP70 and Ki-67 index was demonstrated. A previous study showed the independent prognostic value of Ki-67 index in MBs.23 Patients with MBs exhibiting a Ki-67 index more than 50% differed significantly from patients with MBs exhibiting a Ki-67 index lower than 50% and were associated with worse survival.23 Thus, HSP27 (pSer15) and HSP70 may have prognostic implications in patients with MBs. Apart from that, previous studies in other ma-lignancies have shown that a high level of HSP27 is as-sociated with a diminished cell proliferation rate, while HSP70 overexpression has been shown to correlate with an increased cell proliferation rate, similar to the findings of the present study.33,35 Another important finding was the significantly higher HSP70 expression in large-cell MBs. Large-cell MBs are associated with worse overall survival.20,21 This was also observed in the present study.

Bcl-2 is an antiapoptotic protein known to affect tu-mor cell proliferation. In embryonal brain tumors, bcl-2 expression has been associated with patient prognosis.24 In a previous study, we found that a bcl-2 index over 30% was associated with a worse outcome.24 In the present study, we found a significant positive correlation between HSP70 ex-pression and bcl-2 index. Thus, HSP70 expression deserves further investigation in a larger study for possible prognos-

Fig. 1. A: Heterogeneous immunohistochemical expression of Ki-67 (40%). B: High expression (60%) of p53 in MB. C: High (70%) immunohistochemical expression of bcl-2 protein. Original magnification ×200.

TABLE 2: Expressions of the HSPs, Akt, and phospho-Akt according to MB subtype

ExpressionMB Subtype

Classic (n = 17)* Large-Cell (n = 5)* Extensive Nodularity (n = 2)* Desmoplastic/Nodular (n = 1)

HSP27 (pSer82)† 10.67 ± 17.68 22.71 ± 32.73 0 0HSP27 (pSer15)† 13.97 ± 0.73 13.91 ± 0.92 13.17 ± 0.02 13.16HSP40‡ 2.27 ± 1.3 2.35 ± 0.86 3.68 ± 4.17 8.57HSP60‡ 8.59 ± 8.82 12.13 ± 13.01 71.29 ± 7.21 69.54HSP70‡ 2.63 ± 3.49 7 ± 4.48 6.43 ± 9.1 23.78HSP90-α‡ 3.43 ± 1.83 4.43 ± 0.95 6.37 ± 5.93 9.33Akt‡ 0 0 0 2.63phospho-Akt† 78.5 ± 101.49 89.13 ± 171.75 84.64 ± 119.7 0

* Values are presented as the mean ± SD.† Measured in μ/ml.‡ Measured in ng/ml.

G. A. Alexiou et al.


tic implications. In the present study, there was also a trend toward a positive correlation between HSP40 and p53 ex-pression. Ray et al. have reported that the p53 index was a strong predictor of survival in a large series of 119 patients with MBs.29 However, the exact role of HSP40 and HSP60 requires further elucidation, since these proteins have not been previously investigated in brain tumors.

HSP90 is another antiapoptotic protein, associated with a number of signaling proteins.17 In the present study, HSP90-α protein was selected for investigation, since only the induced HSP90-α and not the constitutive HSP90-b has been found upregulated in glioma.28 Furthermore, HSP90-α has therapeutic implications since both HSP90-α gene silencing and protein inhibitor approaches resulted in a dramatic reduction in cell viability of gliomas.6,27 Alves-pimycin (17-DMAG), an HSP90 inhibitor, is currently in clinical trials for several tumors with favorable results.14,18 In studying MBs, Ayrault et al. reported that, in order for 17-DMAG to exert its antitumorigenic effect, an intact p53 response is required.2 Apart from HSP90, an HSP70 in-hibitor has also been developed and showed promise as an anticancer compound.3 The increased expression of both HSP70 and HSP90-α in the present study suggests a pos-sible therapeutic implication in MB.

In the present study we found activated Akt (phos-phor-Akt) in a substantial number of MBs. Hartmann et al. have reported increased immunohistochemical ex-pression of Ser473-phosphorylated Akt independent of the histopathological subtype, suggesting that PI3K/Akt signaling may play an important role in MBs.12 Further-more, they have reported that PTEN may be involved in the activation of the PI3K/Akt pathway in MBs.12 In the present study no correlation was found between phospho-Akt and MB subtype.

Despite the interesting findings, this study has certain limitations. First, it was performed at a single institution and there was a relatively small number of patients. This might be one of the possible reasons for finding no prog-nostic significance of HSP levels in patients with MBs. Furthermore, we did not perform a molecular analysis to classify MBs into the 4 major groups and to evaluate each tumor subtype with HSP expression. Finally, peripheral blood mononuclear cells of blood samples were used as normal control in our analysis and not normal brain tissue.

ConclusionsThe present study demonstrated a substantial HSP

production in the MB specimens examined. Moreover, to our knowledge, the expression of many various HSPs has not been previously reported in MBs. Taking into con-sideration our findings and the notion that HSPs are an attractive strategy for anticancer therapy, further studies, involving larger series of patients, are obviously neces-sary to clarify the relationship of HSPs with tumor ag-gressiveness and prognosis.

Disclosure

The authors report no conflict of interest concerning the mate-rials or methods used in this study or the findings specified in this paper.

Author contributions to the study and manuscript preparation include the following. Conception and design: Alexiou. Acquisition of data: all authors. Analysis and interpretation of data: Alexiou, Vartholomatos, Stefanaki, Patereli, Dova, Karamoutsios, Lallas, Moschovi, Prodromou. Drafting the article: Alexiou, Stefanaki, Karamoutsios, Prodromou. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Alexiou. Statistical analysis: Alexiou. Administrative/technical/material sup-port: all authors. Study supervision: Alexiou, Stefanaki, Prodromou.

Acknowledgment

The authors would like to thank Mrs. Elissavet Papanikolaou for English-language editing.

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Manuscript submitted February 18, 2013.Accepted July 23, 2013.Please include this information when citing this paper: pub-

lished online August 30, 2013; DOI: 10.3171/2013.7.PEDS1376.Address correspondence to: George Alexiou, M.D., Aetideon 52,

Holargos, Attikis 11561, Greece. email: [email protected].

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Expression of heat shock proteins in medulloblastoma