Stage-dependent expression of PI3K/Akt‑pathway genes in neuroblastoma

  • Authors:
    • Susanne Fransson
    • Frida Abel
    • Per Kogner
    • Tommy Martinsson
    • Katarina Ejeskär
  • View Affiliations

  • Published online on: December 12, 2012     https://doi.org/10.3892/ijo.2012.1732
  • Pages: 609-616
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Abstract

The phosphoinositide-3 kinase (PI3K) pathway plays a critical role in cancer cell growth and survival and has also been implicated in the development of the childhood cancer neuroblastoma. In neuroblastoma high mRNA expression of the PI3K catalytic isoform PIK3CD is associated to favorable disease. Yet, activation of Akt is associated with poor prognosis. Since the contribution of the numerous members of this pathway to neuroblastoma pathogenesis is mainly unknown, genes of the PI3K/Akt pathway were analyzed at the mRNA level through microarrays and quantitative real-time RT-PCR (TaqMan) and at the protein level using western blot analysis. Five genes showed lower mRNA expression in aggressive compared to more favorable neuroblastomas (PRKCZ, PRKCB1, EIF4EBP1, PIK3RI and PIK3CD) while the opposite was seen for PDGFRA. Clustering analysis shows that the expression levels of these six genes can predict aggressive disease. At the protein level, p110δ (encoded by PIK3CD) and p85α isomers (encoded by PIK3R1) were more highly expressed in favorable compared to aggressive neuroblastoma. Evaluation of the expression of these PI3K genes can predict aggressive disease, and indicates stage-dependent involvement of PI3K-pathway members in neuroblastoma.

Introduction

The phosphoinositide-3 kinase (PI3K)/Akt pathway participates in many biological processes such as proliferation, apoptosis, differentiation, metabolism and migration (1). The PI3K signaling cascade is initiated through activation of receptors with intrinsic tyrosine kinase activity, which leads to generation of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3), acting on downstream targets such as PI-dependent kinase (PDK1), integrin-linked kinase (ILK-1) or Akt. Type IA PI3K is a heterodimer composed of a p85 regulatory subunit encoded by PIK3R1, PIK3R2 or PIK3R3 and a p110 catalytic subunit; p110α, p110β or p110δ encoded by PIK3CA, PIK3CB and PIK3CD, respectively. Deregulation of the PI3K/Akt pathway is a recurrent feature in numerous human malignancies with a key role in cancer development, progression and also in resistance to chemotherapy. Over-activity is commonly caused by loss of the tumor suppressor gene PTEN(2,3), oncogenic activation of PIK3CA(4,5) and/or over-stimulation by various growth factors like IGF-1, EGF or VEGF (68).

Neuroblastoma is a pediatric cancer stemming from immature precursors of the sympathetic nervous system with tumors arising in sympathetic ganglia or adrenal gland (9). Neuroblastoma displays high clinical variability, ranging from more favorable stage 1 tumors to highly aggressive stage 4 tumors with many times fatal outcome. The contribution of PI3K/Akt in the carcinogenesis of neuroblastoma is not fully understood. Mutations in the genes PIK3CA and PTEN frequently reported in other malignancies, are rare in neuroblastoma (10,11) although a few mutations have been reported in PIK3CD(12). PIK3CD also show lower expression in aggressive neuroblastomas compared to neuroblastomas with more favorable biology (13,14). Moreover, further connection to the PI3K/Akt pathway is seen through Akt, which is found to be activated in neuroblastoma (15) in an outcome-correlated manner (16). There are several other markers that correlate to grade of disease and/or outcome, such as expression of the different Trk-receptors (17), degree of neural differentiation (18,19) or genetic aberrations such as 1p deletion, 11q deletion, gain of 17q and amplification of the oncogene MYCN(20). PI3K signaling has effect on Mycn protein stability through inactivation of GSK3β and inhibition of PI3K destabilized Mycn and prevented tumor progression in a murine model of neuroblastoma (21).

PI3K inhibition is considered to be one of the most promising targeted therapies for cancer, thus the understanding of the molecular pathology of the individual tumors will be essential to match patients with PI3K inhibitors of differing selectivity profiles. In this study we explored the expression of PI3K/Akt associated genes and found significant differences at both mRNA and protein levels between aggressive and favorable neuroblastoma tumors.

Materials and methods

RNA purification and cDNA preparation

Fresh frozen tumor samples from patients diagnosed with neuroblastoma and staged according to the International Neuroblastoma Staging System Criteria (INSS) and International Neuroblastoma Risk Group (INRG) were used (Table I). Total-RNA was prepared using Totally RNA (Ambion, St. Austin, TX) or RNeasy mini kit (Qiagen, Hilden, Germany) while genomic DNA were removed with DNA-free kit (Ambion). Purity and integrity of the RNA were assayed with spectrophotometer and RNA 6000 Nano Bioanalyzer (Agilent, Palo Alto, CA) before cDNA synthesis using SuperScript™ II Reverse Transcriptase (Invitrogen, Carlsbad, CA).

Table I.

Clinical data.

Table I.

Clinical data.

PatientINSSINRGOutcome1p lossMNA11q lossMethods
QPCRWBArray
18E11LNEDNegNegNeg+
18E51LNEDNegNegNeg+
18E81LNEDNegNegNeg+
19R11LNEDNegNegNeg+
30R91LNEDNegNegNeg+
19R61LDODPosPosNeg+
17E72LNEDNegNegNeg+
10R62LNEDNegNegNeg+
14R92LNEDPosNegNeg+
25R82LNEDNegNegNeg+
27R12LNEDNegNegNeg+
33R72LNEDNegNegNeg+
8E53LNEDNegNegNeg+
16R43LNEDNegPosNeg+
34R53LNEDNegNegNA+
6E93LDODPosNegPos+
13E63LDODPosPosPos+
15E14MNEDPosPosNeg+
10E64MNEDPosPosNeg+
17R34MNEDNegNegNA+
25R34MNEDNegNegNeg+
29R24MNEDPosPosNeg+
32R24MNEDPosNegPos+
40R24MNEDNegNegNeg+
4E14MDODNegNegPos+
3E24MDODNegNegPos+
12E34MDODPosPosNeg+
16E34MDODPosPosNeg+
11E44MDODNegNegPos+
18E44MDODPosPosNeg+
13R04MDODPosPosNeg+
24R34MDODPosPosNA+
26R84MDODPosPosNA+
35R2NALNEDNegNegNeg+
14E61LNEDNegNegNeg++
10R71LNEDNegNegNeg++
35R51LNEDNANANA++
35R81LNEDNegNegNeg++
37R61LNEDNegNegNeg++
26R04MNEDPosPosPos++
25R92LNEDNegNegNeg+++
10R24MDODPosPosNeg+++
15R34MDODPosNegPos+++
34R04MDODNegNegNeg+++
9R93MDODPosNegPos++
15E73LDSCNegNegNeg++
15E33LNEDNegNegNeg++
20R92LNEDNegNegNA++
27R72LNEDNegNegNeg++
25R03LNEDNegNegNeg++
17R24MDODNegNegPos++
28R84MDODNegNegPos++
33R51LNEDNegNegNeg+
13E82LNEDNegNegNeg+
11R43LDODPosPosNeg+
16E94MDODNegPosNeg+
10R83LDODNegNegPos+
39R14MNEDPosPosNeg++
26R91LNEDNegNegNeg+
11E14MNEDNegNegPos+
16E11LNEDNegNegNeg+
23R42LNEDNegNegNeg+
36R3MSMSDODNegNegNeg+

[i] INSS, International Neuroblastoma Staging System; INRG, International Neuroblastoma Risk Group; MNA, MYCN amplification; NA, information not available; UF, unfavorable; F, favorable; L, localized; M, metastasized; MS metastasized stage 4S; NED, no evidence of disease; DOD, dead of disease; DSC, dead by surgical complications; QPCR, quantitative real-time PCR; WB, western blot analysis; Neg, negative; Pos, positive.

Expression analysis by microarray and real-time RT-PCR

Four total-RNAs run on Affymetrix HU133A platform as described previously (46), and another twelve total-RNAs were run on the Affymetrix HU133plus2 platform by Aros Applied Biotechnology AS (www.arosab.com/). Bioconducter for R 2.9.2 (library BioC 2.4) was used to perform gcRMA normalisation for each GeneChip platform set separately. For each probe-set, the maximum expression values over all samples was determined, and probe-sets that showed very low or no detectable expression levels were filtered out (max 2log expression <6). For those probe-sets overlapping the two GeneChip platforms, a probe-specific normalization between the two platforms was performed based on two individuals run on both platforms. Next, the mean log2 expression level for each gene symbol was calculated.

A set of 88 genes with known association to the PI3K/Akt pathway were selected (Table II) and a two-sided t-test was performed to identify genes with significant differential expression when comparing neuroblastoma of low stage (stage 1, 2 and 4S) (n=10) to stage 4 (n=6). Expression of identified genes were verified by quantitative real-time PCR (QPCR) using TaqMan Low Density arrays in a larger set of tumors; stage 1–2 (n=21), stage 4 (n=22) and stage 3 (n=9). Pooled RNA (40 donors) from normal adrenal gland tissue was used as reference (Ambion). QPCR was performed using triplicates with pre-designed primer and probe sets for target genes (PRKCZ: hs.00177051_ml, EIF4EBP1: hs.00607050_ml, PRKZB1: hs.01030676_ml, PDGFRA: hs.00183486_ml, PIK3CD: hs.00192399_ml, PIK3R1: hs.00933163_ml, AKT1: hs.00920503_ml, BAD: hs.00188930_ml, GUSB: hs.99999908_ml) and ABI PRISM® 7900HT Sequence detection system (Applied Biosystems). Quantification was performed using the standard curve method with GUSB (β-glucuronidase) as endogenous control for normalization of gene expression. The logarithms of mean expression levels were used in t-tests of microarray and QPCR data. Expression from microarrays was compared using two-tailed t-test while expression of genes in the validation-set was compared using one-tailed t-test. Statistical calculations and boxplots were made with SPSS ver.18 (SPSS, Chicago, IL) and Excel (Microsoft). Fold change was calculated by dividing the corresponding values for stage 4 with that of stage 1 and 2 neuroblastomas. Unsupervised hierarchal clustering of real-time PCR data from six PI3K pathway genes and 52 primary neuroblastoma samples. The heat map was based on Max linkage.

Table II.

Tested PI3K/Akt associated genes.

Table II.

Tested PI3K/Akt associated genes.

GeneDescriptionGeneDescription
ADARAdenosine deaminase, RNA-specific isoform aMAPK1Mitogen-activated protein kinase 1
AKT1V-akt murine thymoma viral oncogene homolog 1MAPK14Mitogen-activated protein kinase 14
AKT3V-akt murine thymoma viral oncogene homolog 3MAPK3Mitogen-activated protein kinase 3
APCAdenomatous polyposis coliMAPK8Mitogen-activated protein kinase 8
BADBCL2-antagonist of cell death proteinMTCP1Mature T-cell proliferation 1
BTKBruton agammaglobulinemia tyrosine kinaseMYD88Myeloid differentiation primary response gene
CASP9Caspase 9 isoform alpha preproproteinNFKB1Nuclear factor kappa-B, subunit 1
CCND1Cyclin D1NFKBIANuclear factor of kappa light polypeptide gene
CD14CD14 antigen precursorNRASNeuroblastoma RAS viral (v-ras) oncogene
CDC42Small GTP binding protein CDC42PABPC1Poly(A) binding protein, cytoplasmic 1
CDKN1BCyclin-dependent kinase inhibitor 1BPDGFRAPlatelet-derived growth factor receptor alpha
CTMPCarboxyl-terminal modulator proteinPDK13-phosphoinositide dependent protein kinase-1
CHUKConserved helix-loop-helix ubiquitous kinasePDK2Pyruvate dehydrogenase kinase, isozyme 2
CSNK2A1Casein kinase II alpha 1 subunitPIK3CA Phosphoinositide-3-kinase, catalytic, alpha
CTNNB1Catenin (cadherin-associated protein), beta 1PIK3CB Phosphoinositide-3-kinase, catalytic, beta
CUTL1Cut-like homeobox 1PIK3CD Phosphoinositide-3-kinase, catalytic, delta
EIF2AK2Eukaryotic translation initiation factor 2-alphaPIK3CG Phosphoinositide-3-kinase, catalytic, gamma
EIF4A1Eukaryotic translation initiation factor 4APIK3R1 Phosphoinositide-3-kinase, regulatory subunit 1
EIF4BEukaryotic translation initiation factor 4BPIK3R3 Phosphoinositide-3-kinase, regulatory subunit 3
EIF4E2Eukaryotic translation initiation factor 4EPP2AProtein phosphatase 2, catalytic subunit, alpha
EIF4EBP1Eukaryotic translation initiation factor 4EPRKCAProtein kinase C, alpha
EIF4G1Eukaryotic translation initiation factor 4PRKCB1Protein kinase C, beta isoform 1
ELK1ELK1 proteinPRKCZProtein kinase C, zeta
FASLGTumor necrosis factor ligand superfamily member 6PTENPhosphatase and tensin homolog
FKBP1AFK506-binding protein 1APTK2PTK2 protein tyrosine kinase 2
FOSC-fos FBJ murine osteosarcoma viral oncogenePTPN11Protein tyrosine phosphatase, non-receptor type
FOXO1Forkhead box O1RAC1Ras-related C3 botulinum toxin substrate 1
FOXO3Forkhead box O3ARAF1V-raf-1 murine leukemia viral oncogene homolog
FRAP1 (MTOR)FK506 binding protein 12-rapamycin associatedRASA1RAS p21 protein activator 1
GJA1Connexin 43RBL2Retinoblastoma-like 2 (p130)
GRB10Growth factor receptor-bound protein 10RHEBRas homolog enriched in brain
GRB2Growth factor receptor-bound protein 2RHOARas homolog gene family, member A
GSK3BGlycogen synthase kinase 3 betaRPS6KA1Ribosomal protein S6 kinase, 90 kDa, polypeptide
HRASV-Ha-ras Harvey rat sarcoma viral oncogeneRPS6KB1Ribosomal protein S6 kinase, 70 kDa, polypeptide
HSPB1Heat shock 27 kDa protein 1SHC1SHC (Src homology 2 domain containing)
IGF1Insulin-like growth factor 1 iSOS1Son of sevenless homolog 1
IGF1RInsulin-like growth factor 1 receptorSRFSerum response factor
ILKIntegrin-linked kinaseTIRAPToll-interleukin 1 receptor domain-containing
IRAK1Interleukin-1 receptor-associated kinase 1TLR4Toll-like receptor 4
IRS1Insulin receptor substrate 1TOLLIPToll interacting protein
ITGB1Integrin beta 1 isoform 1B precursorTSC1Tuberous sclerosis 1 protein
JUNJun oncogeneTSC2Tuberous sclerosis 2
KRASRas family small GTP binding protein K-RasWASLWiskott-Aldrich syndrome gene-like protein
MAP2K1Mitogen-activated protein kinase kinase 1YWHAHTyrosine 3-monooxygenase/tryptophan
Protein isolation, western blot analysis and antibodies

Fresh frozen neuroblastoma tumors were homogenized using Tissuelyzer (Qiagen) in RIPA lysis buffer supplemented with HALT™ Phosphatase and protease inhibitor cocktail (Pierce, Rockford, IL) while a ready-made protein lysate for normal adrenal gland (20 pooled donors) was purchased from Clontech (Mountain View, CA). SDS-PAGE and western blot analysis were carried out according to standard procedures using 30 μg of total protein lysate. Immunoblotting was performed with rabbit polyclonal antibodies against p85α (no. 06–496) (Millipore, Billerica, MA) 4e-bp1 (no. 9452) (Cell Signaling Technology, Danvers, MA) and PKCβ (sc-209), PKCζ (sc-216), Pdgfrα (sc-338) GAPDH (sc-825778) and p110δ (sc-7176), from Santa Cruz Biotechnology (Santa Cruz, CA). Quantification of proteins was performed with the ImageJ software (available at http://rsb.info.nih.gov/ij). GAPDH was used for normalization in calculation of relative expression. The logarithms of expression levels were calculated and the difference between groups was assessed by a two-tailed independent-samples t-test.

Results

mRNA levels of six PI3K-pathway genes differs between neuroblastoma stages

Analysis of Affymetrix oligo microarray data on a panel of neuroblastoma tumors revealed differential expression between low stage (1, 2 and 4S) and stage 4 patients with statistical significance (p<0.05) for 8 out of 88 genes associated with PI3K/Akt signaling (Table III). Expression of these genes were validated in a larger set of primary neuroblastoma samples using QPCR and the pattern of expression was confirmed for PRKCZ, EIF4EBP1, PRKCB1, PIK3CD, PIK3R1, which showed lower expression in stage 4 compared to stage 1–2 tumors, and PDGFRA, which showed higher expression in stage 4 compared to stage 1–2 tumors (Fig. 1, Table III).

Table III.

Results from microarray and QPCR.

Table III.

Results from microarray and QPCR.

GeneChromosomal localizationMicroarray
QPCR
Fold changeP-value*Fold changeP-value**
PRKCZ1p360.460.020.520.0003
EIF4EBP18p120.600.020.640.006
PRKCB116p110.190.020.280.005
PDGFRA4q1210.400.022.460.01
PIK3CD1p360.310.0010.610.03
PIK3R15q130.440.030.360.03
AKT114q320.770.031.050.43
BAD11q130.460.0040.980.40
GUSB7q11----

* Two-tailed t-test;

** one-tailed t-test.

Clustering of six PI3K-pathway genes

Unsupervised hierarchal clustering using Max linkage of real-time PCR data from PRKCZ, EIF4EBP1, PRKCB1, PIK3CD, PIK3R1 and PDGFRA in 52 primary tumor samples showed that the expression levels of these genes cluster neuroblastomas into metastasizing and localized tumors (Fig. 2).

Low p110δ and p85α protein levels in aggressive neuroblastoma

To further explore the proteins encoded by the differential expressed genes we performed western blot analysis on lysates from 18 primary neuroblastoma tumors and normal adrenal gland. All proteins except 4e-bp1 were detectable in adrenal gland and to various extents in neuroblastoma tumors (Fig. 3A). p110δ (encoded by PIK3CD) was detected in all stages, however overall protein levels of p110δ was significantly lower in stage 4 compared to stage 1–2 neuroblastomas (p=0.04) (Fig. 3B). The overall protein levels of p85α isomers were significantly lower in stage 4 compared to stage 1–2 neuroblastoma (p=0.0015) (Fig. 3C). No other proteins encoded by the genes differently expressed on mRNA-level showed significant differences in protein levels in these 18 tested neuroblastoma protein samples.

Discussion

The PI3K/Akt pathway is central for numerous cellular functions and it is frequently deregulated in human cancers. This pathway is also suggested to be an important player in neuroblastoma development and/or progression and we therefore investigated different actors in PI3K/Akt signaling in primary tumors through analysis at the mRNA and protein level. Five of 88 investigated genes associated to PI3K/Akt signaling pathway showed higher levels of mRNA expression in stage 1–2 neuroblastomas compared to stage 4; EIF4EBP1, PRKCZ, PRKCB1, PIK3RI and PIK3CD. It is notable that the decreased expression of PIK3CD and PRKCZ in stage 4 neuroblastoma may be due to their chromosomal localization at 1p36, a region frequently deleted in stage 4 neuroblastoma.

EIF4EBP1 encodes 4e-bp1, a repressor protein that inhibits the eukaryotic translation initiation factor 4E (eIF4E). High expression of EIF4EBP1 in both favorable and unfavorable neuroblastomas compared to adrenal gland indicates a general upregulation with higher mRNA levels in stage 1–2 compared to stage 4 neuroblastoma (Fig. 1). It is possible that lower expression of EIF4EBP1 mimics the physiological relevance of phosphorylation of 4e-bp1 since both is expected to reduce translational inhibition.

The mRNA expression of PRKCB1 and PRKCZ, encoding PKCβ and PKCζ, respectively, were lower in stage 4 compared to stage 1–2 (Fig. 1). Members of the PKC family have unique and even opposite effects on cell growth, survival and differentiation (2224). PKCβ stimulates growth and proliferation in neuroblastoma (25) although upregulation of both PKCβ and PKCζ was noticed under euxanthone-induced differentiation of a neuroblastoma cell line (26) and PKCβ activation induced apoptosis in HL60-cells (27). PKCζ participate in negative regulation of IRS-1 (28) and have shown proapoptotic functions in ovarian cancer (29). On the other hand, siRNA silencing of PRKCZ impairs migration and invasion in glioblastoma, indicating a role in metastasis (30). This suggests different roles of the PKC isoforms depending on stimuli, and that further effort is needed to elucidate the functions of PRKCZ and PRKCB1 in neuroblastoma.

PDGFRA encodes a cell surface tyrosine kinase receptor important in development of the neural crest and has also been shown to be important in neuroblastoma differentiation (31,32). Moreover, it has also been found to be downregulated during neural differentiation (32). We found PDGFRA to be expressed in all stages even though significantly higher in stage 4 compared to stage 1–2 neuroblastoma, probably explained by the undifferentiated character of all neuroblastomas, especially stage 4. Since PDGFRA also has been found to be mutated or overexpressed in cancer and contribute to cancer development by autocrine or paracrine signaling mechanisms, this could also contribute to the pathogenesis of neuroblastoma (33).

Pten activity can be modulated by the p85 subunit of the PI3K (34,35), which also enhances the phosphatase activity of Pten (36). Consequently, decreased levels of p85 leads to diminished Pten activity and hence increased phosphorylation of Akt. In our material, expression of PIK3R1, encoding three different p85α isomers, was indeed decreased in stage 4 tumors compared to stage 1–2 both on mRNA and on protein level (Figs. 1 and 3). In hepatocellular carcinoma PIK3R1 levels were inversely correlated with grade of malignancy, consistent with reports of tumor suppressing functions of p85 (37,38). Besides modulation of Pten, p85 stabilizes and inhibits the p110α isoform (39) and mutations in the SH2-domain of p85 has been shown to release the inhibitory effect of p110α and leads to constitutive activation of Akt (4042).

Both mRNA and protein levels from PIK3CD/p110δ are decreased in stage 4 neuroblastomas compared to stage 1–2 as described by us and others previously (13,14). Signaling through PI3K is required in neural development (4346) and possibly the δ-isoform could be important in neuroblast differentiation since higher levels of p110δ was detected in stage 1–2 neuroblastoma, commonly expressing more markers of neural differentiation. However, the contribution of the different p110 isoforms in neural differentiation is not fully understood and requires further attention.

Although the molecular mechanisms underlying neuroblastoma are slowly being uncovered, neuroblastoma is still fatal in many cases. In this study we have detected differential expression of several members of the PI3K/Akt pathway on mRNA and/or protein level. Since neuroblastoma is a heterogeneous disease, tumor initiation and progression could occur through activation of different signaling pathways. From the present study we conclude that expression evaluation of a few genes involved in the PI3K-pathway can predict aggressive disease, and our findings indicate a stage-dependent involvement of the PI3K-pathway in neuroblastoma.

Abbreviations:

PI3K

phosphoinositide-3 kinase;

QPCR

quantitative PCR;

INRG

International Neuroblastoma Risk Group;

INSS

International Neuroblastoma Staging System Criteria

Acknowledgements

We thank the Sahlgenska Gothenburg Genomics Core Facility for access to the ABI PRISM®7900HT System and Grissel Faura for technical assistance. This study was supported by grants from the Swedish Cancer Society, the Swedish Children’s Cancer Fund, the Sahlgrenska University Hospital Foundation, the Assar Gabrielsson Foundation, Gunvor and Ivan Svensson’s Foundation, Åke Wiberg’s Foundation, Mary Beves Foundation for research in childhood cancer and Frimurare Barnhusdirektionen.

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February 2013
Volume 42 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

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APA
Fransson, S., Abel, F., Kogner, P., Martinsson, T., & Ejeskär, K. (2013). Stage-dependent expression of PI3K/Akt‑pathway genes in neuroblastoma. International Journal of Oncology, 42, 609-616. https://doi.org/10.3892/ijo.2012.1732
MLA
Fransson, S., Abel, F., Kogner, P., Martinsson, T., Ejeskär, K."Stage-dependent expression of PI3K/Akt‑pathway genes in neuroblastoma". International Journal of Oncology 42.2 (2013): 609-616.
Chicago
Fransson, S., Abel, F., Kogner, P., Martinsson, T., Ejeskär, K."Stage-dependent expression of PI3K/Akt‑pathway genes in neuroblastoma". International Journal of Oncology 42, no. 2 (2013): 609-616. https://doi.org/10.3892/ijo.2012.1732