1 PI3K Pathway Activation Provides a Novel Therapeutic Target for Pediatric

Author Manuscript Published OnlineFirst on September 27, 2013; DOI: 10.1158/1078-0432.CCR-13-0222 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

PI3K pathway activation provides a novel therapeutic target for pediatric ependymoma and is an

independent marker of progression free survival

Hazel A Rogers1, Cerys Mayne1, Rebecca J Chapman1, John-Paul Kilday1, Beth Coyle1, Richard G

Grundy1

1Children’s Brain Tumour Research Centre, D Floor Medical School, Queen’s Medical Centre,

University of Nottingham, Nottingham, NG7 2UH, UK

Running title; Therapeutic potential of PI3K pathway in ependymoma

Corresponding Author; Dr Hazel Rogers, Children’s Brain Tumour Research Centre, D Floor Medical

School, Queen’s Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK

Email; [email protected]

Tel; +44 (0)115 8230718

Fax; +44 (0)115 8230696

Keywords; ependymoma, brain tumor, PI3K pathway, AKT, Cyclin D1

Financial Support; Funding was provided by the Joseph Foote Trust, Gentleman’s Night Out and the

James Tudor Foundation.

Conflict of interest; The authors declare that they have no conflict of interest.

Word count; 4631

5 figures and 1 table

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 27, 2013; DOI: 10.1158/1078-0432.CCR-13-0222 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Translation Relevance

Brain tumors are the leading cause of cancer related deaths in children in the UK. Ependymoma is

the second most common malignant pediatric tumor of the CNS. Currently, there are few effective

adjuvant therapies for pediatric ependymoma, outside confocal radiation, and prognosis remains

poor. The PI3K pathway is one of the most commonly activated pathways in cancer with a number of

drugs targeting the pathway in current clinical trials. Here, we identify the PI3K pathway as a

potential therapeutic target and prognostic marker for ependymoma. We found the PI3K pathway to

be active in 72% of primary pediatric ependymomas suggesting targeting the pathway

therapeutically would be a strategy applicable to a high percentage of patients. Through in vitro

functional analysis we demonstrated a significant link between PI3K pathway activation and cell

proliferation suggesting inhibiting the pathway clinically would reduce ependymoma growth.

Abstract

Purpose: Currently, there are few effective adjuvant therapies for pediatric ependymoma, outside

confocal radiation, and prognosis remains poor. The phosphoinositide 3-kinase (PI3K) pathway is one

of the most commonly activated pathways in cancer. PI3Ks transduce signals from growth factors

and cytokines resulting in the phosphorylation and activation of AKT which in turn induces changes

in cell growth, proliferation and apoptosis.

Experimental Design: PI3K pathway status was analyzed in ependymoma using gene expression data

and immunohistochemical analysis of phosphorylated AKT (P-AKT). The effect of the PI3K pathway

on cell proliferation was investigated by immunohistochemical analysis of cyclin D1 and Ki67, plus in

vitro functional analysis. To identify a potential mechanism of PI3K pathway activation, PTEN protein

expression and the mutation status of PIK3CA was investigated.

Results: Genes in the pathway displayed significantly higher expression in supratentorial compared

to posterior fossa and spinal ependymomas. P-AKT protein expression, indicating pathway

activation, was seen in 72% of tumors (n=169) and P-AKT expression was found to be an

independent marker of a poorer progression free survival. A significant association between PI3K

pathway activation and cell proliferation was identified suggesting pathway activation was

influencing this process. PTEN protein loss was not associated with P-AKT staining and no mutations

were identified in PIK3CA.

Conclusions: Our results suggest the PI3K pathway could act as a biomarker, not only identifying

patients with a worse prognosis, but also those which could be treated with therapies targeted

against the pathway, a strategy potentially effective in a high percentage of ependymoma patients.

Introduction

Ependymoma is the second most common malignant pediatric tumor of the central nervous system

(CNS). Prognosis is relatively poor with a 5 year overall survival rate of 24-82% (1-3). Over half of

cases occur in children under 5 years of age (4-6). 90% of pediatric tumors arise intracranially, 70%

occurring in the posterior fossa, with the remaining 10% in the spinal cord.

Array based studies have identified genetic, epigenetic and transcriptional differences between

tumors arising in different anatomical locations (7-9). However, despite the large amount of

molecular data generated few candidate genes and pathways have been identified that could be

used for targeted therapies. Several prognostic markers have been identified for ependymoma, but

few have been analyzed in sufficient pediatric patients (10). The extent of surgical resection is still

the most consistent marker of outcome (11, 12). However even with complete resection tumor

recurrence occurs in up to 50% of patients (13). A number of putative biological and genomic

prognostic markers have been identified in large cohorts including nucleolin (14), Ki67 (15) 1q gain

(16) nestin (17), Nell2 and Lama2 (18).

The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in

cancer including glioma and medulloblastoma (19, 20). PI3Ks are lipid kinases which transduce

signals from growth factors and cytokines influencing diverse biological functions including cellular

proliferation, survival and motility. In response to upstream signals PI3K phosphorylates the lipid

phosphatidylinositol-,4,5,-bisphosphate which becomes phosphatidyl-3,4,5,-trisphosphate (PIP3)(21).

PIP3 links PI3K to downstream effectors including the serine threonine kinase AKT. When the PI3K

pathway is activated AKT becomes phosphorylated and consequently phosphorylates downstream

targets including GSK3β, FOXO transcription factors and components of the mTOR pathway (22).

In cancer the PI3K pathway is activated through mutations or alterations in many components of the

pathway (23-25). PTEN is a key negative regulator of the pathway and loss of PTEN through mutation

or deletion has been reported in many cancers including glioma (23, 25). Copy number loss of the

PTEN loci has also been seen in ependymoma (7). Activating mutations in the PI3K catalytic subunit

α-isoform gene (PIK3CA) are also found in many common cancers (24).

The high incidence of PI3K pathway activation in cancer has made it an attractive therapeutic target.

Drugs targeting the PI3K pathway have been developed, many of which are being introduced into

clinical trials (22).

We investigated the status of the PI3K pathway in ependymoma. Analysis of mRNA expression array

data and protein expression by immunohistochemistry demonstrated the PI3K pathway to be active

in 72% of primary ependymomas, with a higher percentage of supratentorial (ST) ependymomas

displaying pathway activation than posterior fossa (PF) and spinal (SP) tumors. Pathway activation

was identified as an independent marker of poorer progression free survival. Our data also suggest

PI3K pathway activation induces cell proliferation in ependymoma.

Materials and Methods

Sample information

272 formalin fixed paraffin embedded pediatric ependymoma samples were used, 197 primary and

75 recurrent. Primary and subsequent recurrent samples were used from 30 patients. Samples were

collected from patients diagnosed between 1961 and 2010. The mean and median age of the cohort

was 69 and 46 months respectively. 67% of primary tumors were PF, 22% ST and 6% SP. Data were

unavailable for 9 tumors. 64% of primary tumors were classic and 33% anaplastic ependymoma.

Histopathological information was unavailable for 5 samples. For patients still alive the mean time of

follow up was 89 months and median 83 months. 60% of patients received radiotherapy. Treatment

was unknown for 15% of patients.

Retrospective tumor samples were obtained from the Children’s Cancer and Leukaemia Group

(CCLG) and the Cooperative Human Tissue Network (CHTN). Approximately 50% of the cohort were

samples from patients enrolled in two clinical trials; CNS9204 (26) and CNS9904 (27). 63 primary and

27 recurrent samples were from the CNS9204 trial and 46 primary and 16 recurrent samples from

the CNS9904 trial.

23 snap frozen ependymomas were obtained for DNA extraction, 18 primary and 5 recurrent. For

one patient the primary and two recurrent samples were included. Samples were collected from

patients diagnosed between 1987 and 2007. 56% were PF, 35% ST and 4% SP. For one sample

location was unknown. 61% were classic and 39% anaplastic ependymoma.

19 snap frozen ependymomas were obtained for RNA extraction, 14 primary and 5 recurrent. Two

recurrences were from one patient. Samples were collected from patients diagnosed between 1989

and 2008. 68% were PF, 21% ST and 11% SP. 63% were classic and 37% anaplastic ependymoma. Six

of the tumors used for RNA extraction also had DNA extracted.

Patient clinical information was obtained from CCLG, CHTN, local centres and trial centres. Multiple

Centre Research Ethics (MREC) approval was obtained for the study. Consent for use of tumor

samples was taken in accordance with national tumor banking procedures and the Human Tissue

Act. Work was conducted in premises licensed under the Human Tissue Act.

Expression array analysis

Analysis was undertaken using previously published mRNA expression data (GEO accession number

GSE21687) (7). Raw data were processed using the robust multiarray average (rma) algorithm. Adult

tumors were excluded. Genes differentially expressed between groups (p < 0.05) were identified

using an ANOVA test with a Benjamini and Hochberg multiple test correction (28), using GeneSpring

GX11.0 software (Agilent). Identified gene lists were analyzed using Ingenuity Pathway Analysis (IPA)

9.0 (http://www.ingenuity.com).

Quantitative PCR

RNA was extracted from 40-50 mg of frozen tissue using the mirVanaTM miRNA Isolation kit (Applied

Biosystems). Prior to extraction, a small piece of tissue was prepared on a glass slide as a diagnostic

smear, with subsequent hematoxylin and eosin (H&E) staining, to determine if tumor cells were

present. After extraction RNA was treated with DNase (Promega) (2 U) at 37oC for 30 minutes. cDNA

synthesis was carried out using the Revertaid cDNA synthesis kit (Fermentas) using 500 ng RNA.

Primers were designed to amplify AKT1 (Forward 5’-CTAAGGCCGTGTCTCTGAGG-3’, Reverse 5’-

GAGGGCTTTCCTGTCACAAA-3’, annealing temperature 62oC), PIK3R2 (Forward 5’-

ATGTTGGGGGTCCTAACTCC-3’, Reverse 5’-TGTAAACATGGCAGGTGCAG-3’, annealing temperature

62oC), FGFR3 (Forward 5’-GCCTGGACTGCTACCTTTCA-3’, Reverse 5’-CGTCGCTGGGTTAACAAAAT-3’,

annealing temperature 62oC) and CCND1 (Forward 5’-ACGCTTTGTCTGTCGTGATG-3’, Reverse 5’-

GTGCAACCAGAAATGCACAG-3’, annealing temperature 63oC). GAPDH was used as a housekeeping

gene (Forward 5’-ATGTTCGTCATGGGTGTGAA-3’, Reverse 5’-GTCTTCTGGGTGGCAGTGAT-3’). PCR

reactions were carried out as previously described (8). Relative expression to a calibrator sample

(fetal temporal lobe total RNA, BioChain) was calculated using the Pfafl equation (29).

Immunohistochemistry

Samples were analyzed on tissue microarrays. Following diagnostic pathology review representative

areas of tumor tissue were selected. Three cores from each tumor were included.

Immunohistochemistry (IHC) was carried out as described previously (14). Slides were incubated

with phosphorylated AKT (P-AKT) (ser 473) (1/50, Cell signaling technology), cyclin D1 (1/100,

Abcam), phosphorylated S6 (P-S6) (1/100, Cell signaling technology), Ki67 (1/50, Dako) and PTEN

(1/25, Abcam). P-AKT was scored as positive or negative, with the presence of nuclear staining

noted. For CCND1 the percentage of positive cells was calculated. At least 100 cells per sample were

counted. P-S6 was scored as positive or negative. The Ki67 labeling index was calculated as

previously described (14). PTEN was scored as positive or negative.

Association between clinical factors and immunohistochemical status was investigated using the

Fisher’s Exact Test for categorical variables and T-test for continuous variables. Overall, and

progression free, survival were investigated using the Kaplan Meier method, with differences

estimated using the long-rank (Mantel-Cox) test. Overall survival was defined as the time between

date of diagnosis and date of death and progression free survival as the time between date of

diagnosis and date of first event (recurrence/death). Patients still alive at the end of the study were

censored at the date of last follow up. Multiple factors were analyzed by the Cox proportional

hazard regression model to determine overall survival comparisons with 95% confidence intervals.

Variables with a p-value equal or below 0.25 in univariate analysis were included in multivariate

analysis.

DNA extraction

DNA was extracted from 5–10 mg of frozen tumor tissue as described previously (30). Prior to tissue

extraction, a small piece of tissue was prepared on a glass slide as a diagnostic smear, with

subsequent (H&E) staining, to determine if tumor cells were present.

Sequencing

Primers were designed to amplify exon 9 (Forward 5’-CTGTGAATCCAGAGGGGAAA-3’, Reverse 5’-

CCATTTTAGCACTTACCTGTGAC-3’) and exon 20 (Forward 5’-GTTTCAGGAGATGTGTTACAAGG-3’,

Reverse 5’-TGTTCTTGCTGTAAATTCTAATGCT-3’) of PIK3CA. Standard PCR reactions were carried out.

PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega) and 1.5

U exonuclease I (NEB) at 37oC for 8 min followed by 15 min at 72oC. Sequencing reactions were

carried out by Source Bioscience.

Cell culture

The ependymoma cell line BXD-1425EPN was provided by Dr Xiao-Nan Li, Baylor College of Medicine

and has been previously characterized (31). The cell line was derived from a 9 year old male patient

with a recurrent supratentorial anaplastic ependymoma. The EPN1 cell line was generated and

characterized in house (32). The cell line was derived from a 13 year old male patient with a

recurrent supratentorial ependymoma. The Res196 cell line was provided by Dr DG van Vuurden, VU

University Medical Center, Amsterdam. The line was originally generated and characterized by

Bobola et al. (33) and was derived from a 4 year old male patient with a grade II ependymoma.

Cells were cultured in standard humidified incubators at 5% CO2. BXD-1425EPN and EPN1 lines were

cultured in DMEM (Invitrogen) supplemented with 10% (BXD-1425EPN) or 15% (EPN1) fetal bovine

serum (PAA laboratories) plus antibiotics. Res196 was cultured in DMEM F12 (Invitrogen) with 10%

fetal bovine serum and antibiotics.

MTT assay

Cells were seeded in a 96 well plate and incubated overnight. The following day cells were treated

with Ly294002 (Cell Signaling Technology), BKM120 (Adooq Bioscience) or vehicle control (DMSO).

After 72 hours the level of proliferation was measured using the Cell proliferation kit I (MTT),

following manufacturer’s instructions (Roche). Each condition was performed in triplicate, in three

independent experiments.

Western blot

Cells were seeded in serum free medium and incubated overnight. The following day cells were

treated with Ly294002, BKM120 or vehicle control (DMSO) for 1 hour. For the last 10 minutes EGF

(50 ng/ml, Sigma) was added. Harvested cell pellets were analyzed for protein expression using

western blotting, as previously described (34). Membranes were incubated with primary antibodies

to P-AKT (1 /1000, Cell Signaling Technology), AKT (1/2000, Cell Signaling Technology) and GAPDH

(1/50,000, Abcam).

Results

PI3K pathway was associated with tumor location at the gene expression level

Previous research has shown ependymomas from different locations display distinct biological

profiles. Using published mRNA expression array data (GSE21687) (7), we identified significant

differences in mRNA expression between ependymomas from different locations (PF, ST, SP)

(Supplemental Table 1). Analysis of the top 3000 probes using IPA identified pathways displaying a

significant enrichment in the identified gene list. The top pathway was the PI3K pathway with

multiple genes displaying significantly higher expression in ST tumors (fig. 1a). 73 genes from the

PI3K pathway displayed a significant difference in gene expression between ependymomas from

different locations (Supplemental Table 2, fig 1b). Genes included components of the pathway such

as the AKT genes, growth factors and their receptors, plus downstream genes including cyclin D1

(CCND1).

Four genes were selected for validation using qPCR; AKT1, PIK3R2, FGFR3 and CCND1. PCR was

carried out using an independent cohort of ependymomas to those used in the array analysis. Each

gene displayed higher expression in ST ependymomas compared to PF and SP tumors, agreeing with

the array data (fig 1c).

PI3K pathway activation was linked to prognosis

AKT is a major downstream effector of the PI3K pathway. The protein expression of P-AKT was used

to assess the activation status of the PI3K pathway in a retrospective cohort of 231 ependymomas

(169 primary and 62 recurrent). Positive staining was seen in 122 (72%) primary tumors. Of the

positive samples, 102 (84%) displayed nuclear staining (P-AKT-N) (fig. 2). A significant association

was identified between P-AKT staining and tumor location (p=0.05), with a higher percentage of ST

ependymomas displaying positive staining (84%) than PF (69%) or SP tumors (50%). No association

was found with any other clinical factor. Fifty (81%) recurrences displayed positive staining, 45 with

nuclear staining. Of 24 primary/recurrent pairs analyzed, 19 (79%) displayed positive staining in both

tumors. Three were negative in the primary and positive in the recurrent tumor. One pair were both

negative and one pair displayed positive staining in the primary tumor which was lost in the

recurrent tumor (Supplemental Table 3).

A significant association was identified between P-AKT staining and a worse progression free survival

(PFS) (p=0.049). A more significant result was obtained when only the nuclear positive tumors were

compared to the rest of the cohort (p=0.023) (fig. 2). No association was found with overall survival

(OS). A higher level of significance was seen in multivariate analysis (P-AKT p=0.02, P-AKT-N p=0.011)

(Table 1). Only factors with a p value less than 0.25 in univariate analysis were included in the

multivariate analysis. These factors were; histology; extent of resection; age; ki67, location and

whether patients received radiotherapy (RT) before progression (Supplemental Table 4). The spinal

group was too small to analyze independently. In univariate analysis the PF group displayed the most

significant association with survival compared to the other two locations. Therefore ST and SP

tumors were grouped together and compared to PF tumors for multivariate analysis.

1q status was analyzed in a previous study on a subset of the cohort (n=76, approximately 50% of

the total cohort) (16). P-AKT was found to be independent of 1q status in a multivariate analysis of

the reduced cohort (data not shown). Nucleolin was also analyzed, but did not reach significance in

univariate analysis (data not shown).

A recent publication has divided PF ependymomas into two subgroups which can be identified using

lama2 and nell2 protein expression (18). Lama2 and nell2 protein expression was analyzed in our

cohort using IHC. However, no correlation was found with P-AKT suggesting PI3K pathway activation

was not associated with the PF subgroups (data not shown).

PI3K pathway activation was associated with cyclin D1 expression and cell proliferation

PI3K pathway activation is known to play a role in control of cell proliferation including through

increased expression of cyclin D1. We found cyclin D1 gene expression was significantly higher in ST

tumors (p=0.01) (fig. 3). Cyclin D1 expression also significantly correlated with AKT1 expression

(r=0.588, p=1.6x10-4, n=64). Cyclin D1 protein expression was investigated in our cohort using IHC.

140/161 (87%) primary tumors and 44/50 (88%) recurrences displayed positive staining (fig. 3,

Supplemental Table 3). A significant difference in the percentage of cyclin D1 positive nuclei was

identified between tumors displaying positive or negative P-AKT staining (p=0.003, n=144) (fig. 3).

The correlation with cyclin D1 suggested PI3K pathway activation through AKT maybe influencing cell

proliferation. We therefore looked at the proliferation marker Ki67 using IHC. The percentage of

nuclei displaying positive Ki67 staining ranged from 0-65% in primary tumors (fig. 3). However, Ki67

did not show a significant correlation with P-AKT staining.

To investigate the role of the PI3K pathway in ependymoma functionally we treated ependymoma

cell lines with the PI3K pathway inhibitors Ly294002 and BKM120. Decreased P-AKT protein

expression was seen in cells cultured with inhibitor compared to vehicle only, suggesting the drugs

were specifically inhibiting the PI3K pathway (fig. 4a). A significant decrease in cell proliferation was

seen in drug treated cells after 72 hours (fig. 4b). IC50 concentrations for Ly294002 were 40 μM, 20

μM and 50 μM for BXD-1425EPN, Res196 and EPN1 cell lines respectively. IC50 concentrations for

BKM120 were 2.7 μM, 1.3 μM and 4.4 μM for BXD-1425EPN, Res196 and EPN1 cell lines

respectively. These results suggest the PI3K pathway is increasing cell proliferation in ependymoma.

PI3K pathway activation was not linked to mTOR pathway activation

Activation of the PI3K pathway can induce activation of the mTOR pathway. The mTOR pathway can

also directly regulate the PI3K pathway. mTOR pathway status was investigated in our TMA cohort

using P-S6 to indicate pathway activation. 66/146 (45%) primary and 23/52 (44%) recurrent tumors

displayed positive staining (fig. 3, Supplemental Table 3). No correlation was found between P-AKT

and P-S6 staining suggesting the two pathways were not linked in ependymoma. No association

between P-S6 and OS or PFS was identified.

PI3K pathway activation was not caused by PTEN loss or PIK3CA mutations

PTEN DNA copy number loss has previously been reported in ependymoma, which correlated with

gene expression data (7), suggesting this could be the mechanism of PI3K pathway activation. We

investigated PTEN protein levels using IHC. 10/142 (7%) primary tumors and 2/ 49 (4%) recurrences

were negative (fig. 5 and Supplemental Table 3). PTEN protein expression was not associated with P-

AKT status suggesting PTEN loss was not causing PI3K pathway activation.

Presence of activating mutations in PI3KCA was also investigated. Exons 9 and 20 (mutation

hotspots) were sequenced in DNA extracted from 23 ependymomas. No mutations were found.

Discussion

We have identified PI3K pathway activation in 72% primary pediatric ependymomas suggesting the

pathway is a strong candidate for targeted therapy, likely to be effective in a high proportion of

patients. Our results have also shown pathway status to be an independent marker of poor

prognosis and suggested pathway activation was playing a role in the control of cell proliferation,

which may be acting through cyclin D1.

The results from this study suggest inhibiting the PI3K pathway therapeutically in pediatric

ependymoma is an attractive option, applicable to a high percentage of patients. In a recent drug

screen of a model of a ST subtype of ependymoma, inhibitors of IGF1R, which signals through the

PI3K pathway, and GSK3β were found to disrupt cell proliferation, supporting our conclusions (35). A

number of drugs have been developed against the PI3K pathway, many of which are undergoing

clinical trials, including in adult glioma (22, 36). The IC50 concentrations for BKM120 we found for

ependymoma were similar to those seen in other types of cancer, including adult glioma (37). Phase

I studies in adult tumors have demonstrated that effective plasma concentrations of BKM120 can be

achieved in patients (38). However, phase I studies of BKM120 have not been conducted in pediatric

patients.

We found PI3K pathway status, defined using P-AKT, to be an independent prognostic marker of a

poorer PFS in ependymoma, in comparison to clinical factors and previously reported markers. The

PI3K pathway has also been linked to a poorer prognosis in other cancers including glioma and

breast cancer (19, 39).

Our analysis was undertaken in a retrospective cohort, which included infants and older children and

a variety of treatments. Age at diagnosis and treatment, including extent of resection and

radiotherapy, have previously been linked to prognosis (11, 12, 40-43). The multivariate survival

analysis we undertook demonstrated the association of P-AKT with PFS was independent of these

confounding factors. However, to confirm our result pathway status must be investigated in clinical

trial cohorts. Our cohort contained a subset from patients enrolled on two clinical trials; the

CNS9204 and CNS9904 trials. When these were analyzed independently a significant association with

PFS was only found for P-AKT-N in the CNS9204 trial (Supplemental Figure 1). However p-values

were close to significance in all analyses and suggested the same trend for a worse PFS in patients

with tumors displaying PI3K pathway activation. It is likely that significance was lost due to the lower

number of tumors analyzed in each cohort.

The association with PFS was more significant when tumors displaying nuclear positivity only were

compared to the rest of the cohort. The cellular localization of AKT can determine the downstream

effects of activation (44). Nuclear specific functions for AKT in tumor development and invasion have

been demonstrated in cancer models (45, 46).

If PI3K pathway status is confirmed as a prognostic marker in independent ependymoma cohorts it

could be considered as an attractive marker as it would not only identify patients with a poorer

prognosis, but also those which could be treated with therapies targeted against the pathway.

Our analysis suggested PI3K pathway activation in ependymoma was influencing cell proliferation.

PI3K pathway activation has been linked to cell proliferation in other cancers including

medulloblastoma and glioma (20, 47). Our results suggest the PI3K pathway may be influencing cell

proliferation through cyclin D1. The PI3K pathway has been shown to control cell proliferation

through a number of mechanisms, including regulation of cyclin D1 levels through GSK3β (48, 49).

Two previous studies analyzing cyclin D1 in ependymoma found a lower percentage of positive

tumors than we identified (50, 51). However, both studies analyzed a relatively low number of

patients and included a high percentage of adult tumors. A third study analyzed pediatric cases only

with results closer to our findings (52).

P-AKT expression did not correlate with the proliferation marker Ki67 which might suggest the

change in cyclin D1 levels was not affecting cell proliferation. However, Ki67 is a universal marker of

proliferating cells which can be influenced by many mechanisms that control cell division and is not

directly related to cyclin D1 expression. It may be that cell proliferation is being controlled by

different mechanisms in ependymomas lacking PI3K pathway activation, hence masking any effect

the PI3K pathway may be having. In breast cancer a positive correlation was seen between P-AKT

and cyclin D1 without correlation with Ki67, similar to our results (39). The reduction in cell

proliferation we saw upon PI3K pathway inhibition in the ependymoma cell lines supports our

hypothesis that PI3K pathway activation is increasing cell proliferation in ependymoma.

A significant association between PI3K pathway activation and tumor location was identified at the

gene and protein expression levels. This supports previous research which has identified genomic,

epigenetic and transcriptomic differences between ependymomas arising in different locations (7-9).

However, a relatively high percentage of PF and SP ependymomas displayed PI3K pathway activation

suggesting it also plays an important role in a subset of these tumors. Supratentorial ependymomas

have been associated with a better prognosis than infratentorial tumors, including in the cohort

analyzed in this study. This may seem to contradict the association of PI3K pathway activation with

both supratentorial ependymomas and a poorer PFS. However, our multivariate survival analysis

suggested the association of PI3K pathway activation with PFS was independent of location.

PTEN DNA copy number loss has been shown to occur in ependymoma, which significantly

correlated with PTEN gene expression (7), suggesting it could be the mechanism of PI3K pathway

activation in ependymoma. However, we found no correlation between PTEN protein loss and P-AKT

protein expression suggesting this is not the case. Furthermore, we did not find any mutations in

exons 9 and 20 of PIK3CA, a common mechanism of PI3K pathway activation in cancer (24). It will be

important to determine how the pathway is activated to fully understand what the effects of

inhibiting the pathway therapeutically would be.

Alternative mechanisms of PI3K pathway activation have been identified in other cancers, including

mutations or amplifications of PIK3R1, PDPK1 and AKT1 and 2 (23, 53-56). Analysis of published

ependymoma SNP array data (GSE21687) (7) revealed low copy number gains at AKT1-3 and PDPK1

loci in 10-20% ependymomas but no amplifications. However, no correlation was found between

copy number and gene expression array data.

PI3K pathway activation in cancer has also been shown to be induced through over-expression of

growth factor receptors including EGFR and PDGFR in glioma (57). Previous genomic analysis has

suggested no copy number driven expression of growth factors or their receptors occurs in

ependymoma (7). However, in our analysis we did see differential expression of a number of growth

factor receptor genes, which may be involved in PI3K pathway induction in ependymoma. These

included EGFR, ERBB2, ERBB4 and PDGFRA which have previously been linked to prognosis in

ependymoma in some studies (58-60), but not others (14, 58, 61).

The PI3K pathway can induce activation of the mTOR pathway, which is also deregulated in cancer,

with drugs targeting the pathway in clinical trials for several malignancies (62, 63). We did not find a

correlation between PI3K and mTOR pathway status in ependymoma suggesting the two pathways

were not linked. However, we saw mTOR pathway activation in approximately 50% of ependymomas

suggesting it may be playing an important role in a proportion of tumors. Activation of one or both

of the PI3K and mTOR pathways was seen in 88% of primary ependymoma cases we analyzed

suggesting the majority of pediatric ependymoma patients could be treated with therapies targeting

one of the pathways.

In conclusion we have demonstrated the PI3K pathway to be active in a high percentage of

ependymomas and to be an independent prognostic marker of a worse PFS. Our data suggest

pathway activation in ependymoma increases cell proliferation. The high percentage of patients with

pathway activation and the link to a poor prognosis suggests the potential of targeting the pathway

therapeutically should be investigated. If this proves to be a successful approach it would be

applicable in a high proportion of patients.

Acknowledgements

This was a Children’s Cancer and Leukaemia Group (CCLG) biological study. The authors

acknowledge Professor James Lowe and Professor Keith Robson for histological review and Dr Lisa

Storer and Durgagauri Sabnis for technical support. We thank Dr Xiao-Nan Li, Baylor College of

Medicine for providing the BXD-1425EPN cell line and Dr DG van Vuurden, VU University Medical

Center, Amsterdam for providing the Res196 cell line.

Grant Support

Funding was provided by the Joseph Foote Trust, Gentleman’s Night Out and the James Tudor

Foundation.

References

1. Wright KD, Gajjar A. New chemotherapy strategies and biological agents in the treatment of

childhood ependymoma. Childs Nerv Syst. 2009;25:1275-82.

2. Zacharoulis S, Levy A, Chi SN, Gardner S, Rosenblum M, Miller DC, et al. Outcome for young

children newly diagnosed with ependymoma, treated with intensive induction chemotherapy

followed by myeloablative chemotherapy and autologous stem cell rescue. Pediatr Blood Cancer.

2007;49:34-40.

3. Merchant TE, Li C, Xiong X, Kun LE, Boop FA, Sanford RA. Conformal radiotherapy after

surgery for paediatric ependymoma: a prospective study. Lancet Oncol. 2009;10:258-66.

4. Duffner PK, Krischer JP, Sanford RA, Horowitz ME, Burger PC, Cohen ME, et al. Prognostic

factors in infants and very young children with intracranial ependymomas. Pediatr Neurosurg.

1998;28:215-22.

5. Amirian ES, Armstrong TS, Aldape KD, Gilbert MR, Scheurer ME. Predictors of survival among

pediatric and adult ependymoma cases: a study using Surveillance, Epidemiology, and End Results

data from 1973 to 2007. Neuroepidemiology. 2012;39:116-24.

6. McGuire CS, Sainani KL, Fisher PG. Both location and age predict survival in ependymoma: a

SEER study. Pediatr Blood Cancer. 2009;52:65-9.

7. Johnson RA, Wright KD, Poppleton H, Mohankumar KM, Finkelstein D, Pounds SB, et al.

Cross-species genomics matches driver mutations and cell compartments to model ependymoma.

Nature. 2010;466:632-6.

8. Rogers HA, Kilday JP, Mayne C, Ward J, Adamowicz-Brice M, Schwalbe EC, et al.

Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which

includes the loss of tumor suppressor genes involved in the control of cell growth and death. Acta

Neuropathol. 2012;123:711-25.

9. Taylor MD, Poppleton H, Fuller C, Su X, Liu Y, Jensen P, et al. Radial glia cells are candidate

stem cells of ependymoma. Cancer Cell. 2005;8:323-35.

10. Kilday JP, Rahman R, Dyer S, Ridley L, Lowe J, Coyle B, et al. Pediatric ependymoma:

biological perspectives. Mol Cancer Res. 2009;7:765-86.

11. Tihan T, Zhou T, Holmes E, Burger PC, Ozuysal S, Rushing EJ. The prognostic value of

histological grading of posterior fossa ependymomas in children: a Children's Oncology Group study

and a review of prognostic factors. Mod Pathol. 2008;21:165-77.

12. Horn B, Heideman R, Geyer R, Pollack I, Packer R, Goldwein J, et al. A multi-institutional

retrospective study of intracranial ependymoma in children: identification of risk factors. J Pediatr

Hematol Oncol. 1999;21:203-11.

13. Sowar K, Straessle J, Donson AM, Handler M, Foreman NK. Predicting which children are at

risk for ependymoma relapse. J Neurooncol. 2006;78:41-6.

14. Ridley L, Rahman R, Brundler MA, Ellison D, Lowe J, Robson K, et al. Multifactorial analysis of

predictors of outcome in pediatric intracranial ependymoma. Neuro Oncol. 2008;10:675-89.

15. Bennetto L, Foreman N, Harding B, Hayward R, Ironside J, Love S, et al. Ki-67

immunolabelling index is a prognostic indicator in childhood posterior fossa ependymomas.

Neuropathol Appl Neurobiol. 1998;24:434-40.

16. Kilday JP, Mitra B, Domerg C, Ward J, Andreiuolo F, Osteso-Ibanez T, et al. Copy number gain

of 1q25 predicts poor progression-free survival for pediatric intracranial ependymomas and enables

patient risk stratification: a prospective European clinical trial cohort analysis on behalf of the

Children's Cancer Leukaemia Group (CCLG), Societe Francaise d'Oncologie Pediatrique (SFOP), and

International Society for Pediatric Oncology (SIOP). Clin Cancer Res. 2012;18:2001-11.

17. Milde T, Hielscher T, Witt H, Kool M, Mack SC, Deubzer HE, et al. Nestin expression identifies

ependymoma patients with poor outcome. Brain Pathol. 2012;22:848-60.

18. Witt H, Mack SC, Ryzhova M, Bender S, Sill M, Isserlin R, et al. Delineation of two clinically

and molecularly distinct subgroups of posterior fossa ependymoma. Cancer Cell. 2011;20:143-57.

19. Chakravarti A, Zhai G, Suzuki Y, Sarkesh S, Black PM, Muzikansky A, et al. The prognostic

significance of phosphatidylinositol 3-kinase pathway activation in human gliomas. J Clin Oncol.

2004;22:1926-33.

20. Hartmann W, Digon-Sontgerath B, Koch A, Waha A, Endl E, Dani I, et al. Phosphatidylinositol

3'-kinase/AKT signaling is activated in medulloblastoma cell proliferation and is associated with

reduced expression of PTEN. Clin Cancer Res. 2006;12:3019-27.

21. Vogt PK, Bader AG, Kang S. Phosphoinositide 3-kinase: from viral oncoprotein to drug target.

Virology. 2006;344:131-8.

22. Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide 3-kinase pathway in

cancer. Nat Rev Drug Discov. 2009;8:627-44.

23. Comprehensive genomic characterization defines human glioblastoma genes and core

pathways. Nature. 2008;455:1061-8.

24. Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High frequency of mutations

of the PIK3CA gene in human cancers. Science. 2004;304:554.

25. Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, et al. High-

throughput oncogene mutation profiling in human cancer. Nat Genet. 2007;39:347-51.

26. Grundy RG, Wilne SA, Weston CL, Robinson K, Lashford LS, Ironside J, et al. Primary

postoperative chemotherapy without radiotherapy for intracranial ependymoma in children: the

UKCCSG/SIOP prospective study. Lancet Oncol. 2007;8:696-705.

27. Grundy R, Jaspan T, Macarthur D, Wilne S, Kilday JP, Mallucci C, et al. Outcome of children

treated for intracranial ependymoma: the first SIOP trial. Pediatr Blood Cancer. 2012;59:992.

28. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate - a Practical and Powerful

Approach to Multiple Testing. Journal of the Royal Statistical Society Series B-Methodological.

1995;57:289-300.

29. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR.

Nucleic Acids Res. 2001;29:e45.

30. Rogers HA, Miller S, Lowe J, Brundler MA, Coyle B, Grundy RG. An investigation of WNT

pathway activation and association with survival in central nervous system primitive

neuroectodermal tumours (CNS PNET). Br J Cancer. 2009;100:1292-302.

31. Yu L, Baxter PA, Voicu H, Gurusiddappa S, Zhao Y, Adesina A, et al. A clinically relevant

orthotopic xenograft model of ependymoma that maintains the genomic signature of the primary

tumor and preserves cancer stem cells in vivo. Neuro Oncol. 2010;12:580-94.

32. Hussein D, Punjaruk W, Storer LC, Shaw L, Othman R, Peet A, et al. Pediatric brain tumor

cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion. Neuro Oncol.

2011;13:70-83.

33. Bobola MS, Silber JR, Ellenbogen RG, Geyer JR, Blank A, Goff RD. O6-methylguanine-DNA

methyltransferase, O6-benzylguanine, and resistance to clinical alkylators in pediatric primary brain

tumor cell lines. Clin Cancer Res. 2005;11:2747-55.

34. Levesley J, Lusher ME, Lindsey JC, Clifford SC, Grundy R, Coyle B. RASSF1A and the BH3-only

mimetic ABT-737 promote apoptosis in pediatric medulloblastoma cell lines. Neuro Oncol.

2011;13:1265-76.

35. Atkinson JM, Shelat AA, Carcaboso AM, Kranenburg TA, Arnold LA, Boulos N, et al. An

integrated in vitro and in vivo high-throughput screen identifies treatment leads for ependymoma.

Cancer Cell. 2011;20:384-99.

36. Wen PY, Lee EQ, Reardon DA, Ligon KL, Alfred Yung WK. Current clinical development of PI3K

pathway inhibitors in glioblastoma. Neuro Oncol. 2012;14:819-29.

37. Koul D, Fu J, Shen R, LaFortune TA, Wang S, Tiao N, et al. Antitumor activity of NVP-BKM120-

-a selective pan class I PI3 kinase inhibitor showed differential forms of cell death based on p53

status of glioma cells. Clin Cancer Res. 2012;18:184-95.

38. Bendell JC, Rodon J, Burris HA, de Jonge M, Verweij J, Birle D, et al. Phase I, dose-escalation

study of BKM120, an oral pan-Class I PI3K inhibitor, in patients with advanced solid tumors. J Clin

Oncol. 2012;30:282-90.

39. Capodanno A, Camerini A, Orlandini C, Baldini E, Resta ML, Bevilacqua G, et al. Dysregulated

PI3K/Akt/PTEN pathway is a marker of a short disease-free survival in node-negative breast

carcinoma. Hum Pathol. 2009;40:1408-17.

40. Pejavar S, Polley MY, Rosenberg-Wohl S, Chennupati S, Prados MD, Berger MS, et al.

Pediatric intracranial ependymoma: the roles of surgery, radiation and chemotherapy. J Neurooncol.

2012;106:367-75.

41. Perilongo G, Massimino M, Sotti G, Belfontali T, Masiero L, Rigobello L, et al. Analyses of

prognostic factors in a retrospective review of 92 children with ependymoma: Italian Pediatric

Neuro-oncology Group. Med Pediatr Oncol. 1997;29:79-85.

42. Sala F, Talacchi A, Mazza C, Prisco R, Ghimenton C, Bricolo A. Prognostic factors in childhood

intracranial ependymomas: the role of age and tumor location. Pediatr Neurosurg. 1998;28:135-42.

43. Vaidya K, Smee R, Williams JR. Prognostic factors and treatment options for paediatric

ependymomas. Journal of clinical neuroscience : official journal of the Neurosurgical Society of

Australasia. 2012;19:1228-35.

44. Saji M, Ringel MD. The PI3K-Akt-mTOR pathway in initiation and progression of thyroid

tumors. Mol Cell Endocrinol. 2010;321:20-8.

45. Saji M, Vasko V, Kada F, Allbritton EH, Burman KD, Ringel MD. Akt1 contains a functional

leucine-rich nuclear export sequence. Biochem Biophys Res Commun. 2005;332:167-73.

46. Trotman LC, Alimonti A, Scaglioni PP, Koutcher JA, Cordon-Cardo C, Pandolfi PP.

Identification of a tumour suppressor network opposing nuclear Akt function. Nature. 2006;441:523-

47. Li DM, Sun H. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle

arrest in human glioblastoma cells. Proc Natl Acad Sci U S A. 1998;95:15406-11.

48. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators

of growth and metabolism. Nat Rev Genet. 2006;7:606-19.

49. Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3beta regulates cyclin D1

proteolysis and subcellular localization. Genes Dev. 1998;12:3499-511.

50. Prayson RA. Cyclin D1 and MIB-1 immunohistochemistry in ependymomas: a study of 41

cases. American journal of clinical pathology. 1998;110:629-34.

51. Zawrocki A, Izycka-Swieszewska E, Papierz W, Liberski PP, Zakrzewski K, Biernat W. Analysis

of the prognostic significance of selected morphological and immunohistochemical markers in

ependymomas, with literature review. Folia Neuropathol. 2011;49:94-102.

52. Zamecnik J, Snuderl M, Eckschlager T, Chanova M, Hladikova M, Tichy M, et al. Pediatric

intracranial ependymomas: prognostic relevance of histological, immunohistochemical, and flow

cytometric factors. Mod Pathol. 2003;16:980-91.

53. Brugge J, Hung MC, Mills GB. A new mutational AKTivation in the PI3K pathway. Cancer Cell.

2007;12:104-7.

54. Carpten JD, Faber AL, Horn C, Donoho GP, Briggs SL, Robbins CM, et al. A transforming

mutation in the pleckstrin homology domain of AKT1 in cancer. Nature. 2007;448:439-44.

55. Ruggeri BA, Huang L, Wood M, Cheng JQ, Testa JR. Amplification and overexpression of the

AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Mol Carcinog. 1998;21:81-

56. Staal SP. Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2:

amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci U S A.

1987;84:5034-7.

57. Nazarenko I, Hede SM, He X, Hedren A, Thompson J, Lindstrom MS, et al. PDGF and PDGF

receptors in glioma. Ups J Med Sci. 2012;117:99-112.

58. Gilbertson RJ, Bentley L, Hernan R, Junttila TT, Frank AJ, Haapasalo H, et al. ERBB receptor

signaling promotes ependymoma cell proliferation and represents a potential novel therapeutic

target for this disease. Clin Cancer Res. 2002;8:3054-64.

59. Mendrzyk F, Korshunov A, Benner A, Toedt G, Pfister S, Radlwimmer B, et al. Identification of

gains on 1q and epidermal growth factor receptor overexpression as independent prognostic

markers in intracranial ependymoma. Clin Cancer Res. 2006;12:2070-9.

60. Moreno L, Popov S, Jury A, Al Sarraj S, Jones C, Zacharoulis S. Role of platelet derived growth

factor receptor (PDGFR) over-expression and angiogenesis in ependymoma. J Neurooncol. 2012.

61. Modena P, Buttarelli FR, Miceli R, Piccinin E, Baldi C, Antonelli M, et al. Predictors of

outcome in an AIEOP series of childhood ependymomas: a multifactorial analysis. Neuro Oncol.

2012;14:1346-56.

62. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274-

93.

63. Wander SA, Hennessy BT, Slingerland JM. Next-generation mTOR inhibitors in clinical

oncology: how pathway complexity informs therapeutic strategy. J Clin Invest. 2011;121:1231-41.

Table 1 Multivariate survival analysis

Factor Significance Hazard 95% Confidence Ratio Interval P-AKT (positive vs negative) 0.020a 2.060 1.120-3.788 Histology (ependymoma vs anaplastic ependymoma) 0.617 0.873 0.512-1.487 Resection (incomplete vs complete) 3.0x10-4b 2.65 1.565-4.487 Location (PF vs ST+SP) 0.088 1.784 0.917-3.470 Age (continuous variable) 0.540 1.002 0.995-1.009 Ki67 (continuous variable) 0.111 1.061 0.986-1.142 Radiotherapy (yes vs no) 0.129 0.618 0.332-1.150 P-AKT-N (nuclear vs cytoplasmic or negative) 0.011a 2.012 1.179-3.456 Histology (ependymoma vs anaplastic ependymoma) 0.546 0.849 0.501-1.442 Resection (incomplete vs complete) 0.001b 2.518 1.494-4.243 Location (PF vs ST+SP) 0.099 1.744 0.901-3.373 Age (continuous variable) 0.628 1.002 0.995-1.009 Ki67 (continuous variable) 0.078 1.067 0.993-1.147 Radiotherapy (yes vs no) 0.176 0.652 0.351-1.212 ap < 0.05, b p < 0.01

Figure Legends

Fig. 1

Analysis of genes displaying significant differences in mRNA expression between ependymomas from

different locations (PF, ST, SP), using IPA, identified pathways significantly enriched. The top

pathway was the PI3K pathway (a). Genes highlighted in green were up-regulated in ST tumors and

genes in pink up-regulated in PF tumors. The majority of genes displayed higher expression in ST

tumors. Hierarchical clustering of PI3K genes displaying significant differences segregated

ependymomas according to location (b). Analysis was undertaken using previously published

expression array data (Johnson et al. 2010, GSE21687). The dendogram displayed along the

horizontal axis represents the clustering of the ependymoma tumors. The dendogram displayed

along the vertical axis represents the clustering of the genes. (c) Validation of AKT1, PIK3R2, FGFR2

and CCND1 gene expression using qPCR. Each gene displayed higher expression in ST ependymomas

compared to PF and SP tumors, agreeing with the array data. Expression is displayed relative to fetal

temporal lobe.

Fig. 2

Three patterns of P-AKT protein expression were seen in ependymoma, measured by IHC, negative

(a), cytoplasmic (b) and nuclear (c). (d) In univariate survival analysis a significant association with a

worse PFS was found for both P-AKT and P-AKT-N.

Fig. 3

Cyclin D1 gene expression was significantly higher in ST ependymomas compared to PF and SP

tumors (a) (ANOVA p = 0.01). The percentage of nuclei displaying cyclin D1 protein expression was

significantly higher in tumors displaying PI3K pathway activation, defined by P-AKT (b) (t-test p =

0.003). (c) displays an example of cyclin D1 protein expression in an ependymoma. No correlation

was seen between ki67 protein expression (d) and PI3K pathway activation. mTOR pathway

activation, measured by P-S6 protein expression (e), was seen in 45% primary ependymomas but did

not correlate with PI3K pathway activation.

Fig. 4

Ependymoma cell lines were treated with indicated concentrations of Ly294002 and BKM120 for 1

hour, with EGF (50 ng/ml) stimulation for the last 10 minutes. Loss of P-AKT was seen with increasing

concentrations of each inhibitor (a). Treatment with the inhibitors resulted in a decrease in cell

growth. Cells were incubated with indicated concentrations of the two inhibitors for 72 hours. Cell

growth was measured using an MTT assay. A significant decrease in proliferation was seen in drug

treated cells compared to vehicle control (b). * P<0.05, ** P<0.001

Fig. 5

PTEN protein expression was measured using IHC. Loss of expression (a) was seen in 7% of primary

tumors and positive expression (b) was seen in the remaining 93%.

PI3K pathway activation provides a novel therapeutic target for pediatric ependymoma and is an independent marker of progression free survival

Hazel A Rogers, Cerys Mayne, Rebecca J Chapman, et al.

Clin Cancer Res Published OnlineFirst September 27, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-0222

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2013/09/24/1078-0432.CCR-13-0222.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2013/09/24/1078-0432.CCR-13-0222. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.