research article · figure 1. otx2 is amplified in medulloblastoma cell line d458med. a,...

Identification of OTX2 as a Medulloblastoma Oncogene Whose

Product can be Targeted by All-Trans Retinoic Acid

Chunhui Di,1Shaoxi Liao,

1David C. Adamson,

1Timothy J. Parrett,

1Daniel K. Broderick,

1Qun Shi,

1

Christoph Lengauer,2Jordan M. Cummins,

2Victor E. Velculescu,

2Daniel W. Fults,

3

Roger E. McLendon,1Darell D. Bigner,

1and Hai Yan

1

1Brain Tumor Center, Department of Pathology, Duke University Medical Center, Durham, North Carolina; 2Johns Hopkins UniversityMedical Institutions, Baltimore, Maryland; and 3University of Utah School of Medicine, Salt Lake City, Utah

Abstract

Through digital karyotyping of permanent medulloblastomacell lines, we found that the homeobox gene OTX2 wasamplified more than 10-fold in three cell lines. Geneexpression analyses showed that OTX2 transcripts werepresent at high levels in 14 of 15 (93%) medulloblastomaswith anaplastic histopathologic features. Knockdown of OTX2expression by siRNAs inhibited medulloblastoma cell growthin vitro , whereas pharmacologic doses of all-trans retinoicacid repressed OTX2 expression and induced apoptosis only inmedulloblastoma cell lines that expressed OTX2. Theseobservations suggest that OTX2 is essential for the pathogen-esis of anaplastic medulloblastomas and that these tumorsmay be amenable to therapy with all-trans-retinoic acid.(Cancer Res 2005;65(3): 919-24)

Introduction

Primary brain tumors are a leading cause of cancer death inchildren, andmedulloblastoma is the most frequentmalignant braintumor in this age group. Despite therapeutic advances, more thanone third of children with medulloblastoma die from the diseasewithin 5 years of diagnosis, and the remaining survivors experiencesignificant toxicities from extant therapies (1, 2). Elucidation of themolecular pathogenesis of medulloblastoma may suggest noveltherapeutic targets. Whereas comprehensive screens for activatingor inactivating mutations would require sequencing and functionalstudies of tens of thousands of genes, measurements of the genomicDNA copy number, or gene dosage, of chromosomal segments is farmore amenable to analysis. Complete sequencing of the humangenome hasmade possible the development of novel techniques thatnarrow the resolving power of genome-wide screens to regionscovering one or a small handful of genes (V1 Mb; refs. 3, 4). Withdigital karyotyping and gene expression analysis, we identifiedgenomic amplification and overexpression of OTX2 gene inmedulloblastomas. Medulloblastoma cell lines overexpressingOTX2 were growth inhibited by pharmacologic doses of all-trans-retinoic acid (ATRA) and knockdown of OTX2 expression by siRNA.

Materials and Methods

Tissue Samples. Brain tumor cell lines and frozen primary tumor

samples were obtained from the Duke University Brain Tumor Center

Tissue Bank. Acquisition of tissue specimens was approved by the Duke

University Health System Institutional Review Board and was done inaccordance with the Health Insurance Portability and Accountability Act of

1996 regulations.

Digital Karyotyping. Digital karyotyping libraries were constructed as

previously described (3, 5). Briefly genetic tags were matched to the humangenome, and tag densities were evaluated by using a digital karyotyping

software package. Genomic densities were calculated as the ratio of

experimental tags to the number of virtual tags present in a fixed window.

Sliding windows of 200 virtual tags in size were used to identify regions ofincreased and decreased genomic density. Chromosomal regions were

considered amplified if maximal genomic densities were >6 genome copies

per haploid genome. Digital karyotyping protocols and software forextraction and analysis of genomic tags are available at http://www.digi-

talkaryotyping.org.

Serial Analysis of Gene Expression Data Analysis. The serial analysis

of gene expression (SAGE) data were obtained from the National Center forBiotechnology Information Cancer Genome Anatomy Project repository

(http://cgap.nci.nih.gov/SAGE). The presence of OTX2 SAGE tags in a total

of 157 tumors and 54 normal human tissues was identified by using the

SAGE Anatomic Viewer.Quantitative Real-time PCR. Genomic DNA content differences of

OTX2 or C14orf101 between medulloblastoma cells and normal cells were

determined by using the quantitative real-time PCR (Q-PCR) method as

previously described (5). cDNA from medulloblastoma samples was used tomeasure the level of OTX2 mRNA expression. cDNA from normal human

cerebellum was used as the normal control, and cDNA content was

normalized to that of GAPDH .Cell Proliferation Assay. Cell viability was determined by the 3-[4,5-

dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide MTT assay.

siRNA-Mediated ‘‘Knockdown’’ of OTX2 Expression. To target

OTX2 , siRNA#1 GGAGGUGGCACUGAAAAUCtt and siRNA#2 GGACA-CUAAUUCAUCUGUAtt (Ambion, Austin, TX) were generated using OTX2

sequences. Cells were collected at 48 hours after siRNA transfection to

generate cDNA for Q-PCR. After three doubling times, the cells were

assayed for viability by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetra-zolium bromide MTT assay.

Colony Formation in Soft Agar Assay. The cells were plated at 5 � 103

in triplicates in 0.5% agarose-coated 24-well plates with or without ATRA(Sigma, St. Louis, MO). After 2 weeks, the number of colonies was counted.

Apoptosis. Cell apoptosis induced by ATRA was determined by the Cell

Death Detection ELISAplus method (Roche Diagnostic Co., Indianapolis, IN).

Results and Discussion

We initially used digital karyotyping to analyze quantitativelythe genomic elements at high resolution in the medulloblastomacell line D458MED (3). A digital karyotyping library of 179,299genomic tags from the D458MED cell line was generated,permitting analysis of loci distributed at an average distance of33 kb throughout the genome. Analysis of the tag densitiesrevealed subchromosomal regions of amplification and deletioncommonly associated with medulloblastomas (ref. 6; Fig. 1A),

Note: C. Di, S. Liao, and D. Adamson contributed equally to this work.Requests for reprints: Hai Yan, Duke University Medical Center, Department of

Pathology, DUMC-3156, 199A-MSRB Building, Research Drive, Durham, NC 27710.Phone: 919-668-7850; Fax: 919-684-8756; E-mail: [email protected].

I2005 American Association for Cancer Research.

www.aacrjournals.org 919 Cancer Res 2005; 65:(3). February 1, 2005

Research Article

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including amplification of the C-MYC oncogene on chromosome8q24.21, loss of chromosome 17p, and gain of chromosome 17q.Importantly, subtle changes below the level of resolutionachievable with traditional measurements were also identified.Most striking was a 28-fold amplification located at base pairs55,051,299-55,437,589 (UCSC human genome assembly, July 2003freeze) on chromosome 14q22.3. Examination of a public humangenome database (http://genome.ucsc.edu/cgi-bin/hgGateway)identified one known and one predicted gene in the amplifiedsegment, full-length OTX2 (55,257,468-55,267,225) and a COOH-terminal portion of C14orf101 (55,036,584-55,105,043), respectively(Fig. 1B). Amplification of 20 copies per haploid genome wasconfirmed with Q-PCR analysis of genomic DNA using multiplesets of primers specific to the genomic sequences of OTX2 andC14orf101 (data not shown).After Q-PCR evaluation of genomic DNA from 12 additional

medulloblastoma cell lines and 44 clinical medulloblastomasamples, C14orf101 amplification was observed in cell lineD487MED, and marked OTX2 amplification was identified in bothD487MED and D425MED (10- and 25-fold, respectively). Notably,cell line D425MED was derived from a patient with a primarycerebellar medulloblastoma, whereas D458MED, the cell line used

for the original digital karyotyping, was established from cerebro-spinal fluid samples of the same patient at a later date, indicatingthat the OTX2 amplification occurred before the tumor metasta-sized and was maintained by the metastasized cells. We furtherassessed OTX2 amplification by fluorescence in situ hybridization.Increased OTX2 copy number was observed in D425MED,D458MED, and D487MED (data not shown).OTX2 amplification is particularly interesting because of OTX2 ’s

role in normal cerebellar development (7). OTX2 is a member of ahighly conserved family containing the bicoid-like homeodomaintranscription factors that control the developmental programsunderlying brain morphogenesis (8). In the embryo, OTX2 isnormally expressed throughout the posterior cerebellum, withinthe external granular layer, and in the emerging internal granularlayer and then disappears in later life (9). Elimination of OTX2function in the mouse results in defective development of therostral neuroectoderm, leading to a headless phenotype (10),whereas mice with induced ectopic OTX2 expression in theanterior hindbrain display cerebellar ataxia (11). Several develop-mentally related and hindbrain-specific mRNA transcripts havebeen found at abnormally high levels in medulloblastomas by SAGEand suppression subtraction hybridization (12, 13), supporting the

Figure 1. OTX2 is amplified in medulloblastoma cell line D458MED. A, low-resolution tag density maps of digital karyotyping reveal subchromosomal changes inD458MED. For all chromosomes (labels 1-22 , x , and y ), values on the y -axis indicate genome copies per haploid genome, and values on the x -axis representpositions along the chromosome in Mb. Two prominent peaks, which contain large-scale genomic amplifications, are clearly revealed on chromosomes 8 and 14.Loss of heterozygosity of 17p and gain of 17q is also observed. B, high-resolution tag density maps identify OTX2 amplification on chromosome 14q22.3. An enlargedview of the region of amplification on chromosome 14 was determined from sliding windows of 200 virtual tags. Genes present within the region are indicated belowon a high-resolution map, displaying that the OTX2 gene was entirely contained in the amplified region.

Cancer Research

Cancer Res 2005; 65:(3). February 1, 2005 920 www.aacrjournals.org



hypothesis that these tumors originate from dysregulation of thedevelopmental programs in cerebellar granular cells or theirprecursor. OTX2 has been identified as one of these genes.However, as with the other genes identified in prior studies, thisrelationship was associative and not proof of causation. Thefinding of highly specific and marked OTX2 genetic amplificationsin a subset of the medulloblastomas implies that OTX2 is atargeted oncogene in medulloblastoma pathogenesis and notmerely transcriptionally upregulated by other oncogenic events.In order to examine OTX2 expression, we evaluated OTX2

transcript levels by SAGE data mining 211 libraries obtained fromnormal human tissues and tumors (http://cgap.nci.nih.gov/SAGE;ref. 14). The data revealed a dramatic overexpression of OTX2 inmedulloblastomas but not in any other tumor type or normal

Table 1. Presentation of OTX2 transcript in SAGElibraries

Library No.

SAGE

libraries

No.

libraries

containingOTX2 tag

No. total

OTX2

tagobserved

Average

no. of

OTX2tags/

library

Medullo-

blastoma

24 18 753 31.38

Breastcarcinoma

27 2 6 0.22

Astrocytoma 36 2 6 0.17

Ependymoma 12 0 0 0

Meningioma 6 0 0 0Oligoden-

droglioma

2 0 0 0

Thyroidfollicular

carcinoma

1 0 0 0

Lung adeno-

carcinoma

2 0 0 0

Stomach

carcinoma

4 0 0 0

Pancreas

carcinoma

6 0 0 0

Liver

cholangio-

carcinoma

3 0 0 0

Peritoneummesothelioma

1 0 0 0

Kidney

carcinoma

1 0 0 0

Colon

adenocar-

cinoma

6 0 0 0

Ovaryadenocar-

cinoma

5 0 0 0

Prostate

carcinoma

11 0 0 0

Cartilage

chondro-

sarcoma

8 0 0 0

Skin

melanoma

2 0 0 0

Normal

thalamus

1 1 8 8

Normal

retina

4 3 7 1.75

Normal

cerebellum

2 0 0 0

Normal

cortex

3 0 0 0

Normalthyroid

1 0 0 0

Normal

lung

3 0 0 0

Normalheart

1 0 0 0

Normal

breast

9 0 0 0

(Continued)

Table 1. Presentation of OTX2 transcript in SAGElibraries (Cont’d)

Library No.

SAGE

libraries

No.

libraries

containingOTX2 tag

No. total

OTX2

tagobserved

Average

no. of

OTX2tags/

library

Normal

stomach

2 0 0 0

Normalpancreas

3 0 0 0

Normal

liver

1 0 0 0

Normalkidney

3 0 0 0

Normal

colon

2 0 0 0

Normal

peritoneum

1 0 0 0

Normal

spinal cord

1 0 0 0

Normal

ovary

2 0 0 0

Normal

placenta

2 0 0 0

Normal

prostate

4 0 0 0

Normal

bonemarrow

3 0 0 0

Normal

muscle

2 0 0 0

Normal

skin

1 0 0 0

Normal

lymph node

1 0 0 0

Normal

leukocytes

2 0 0 0

NOTE: SAGE analysis of 157 tumors and 54 normal human tissues

revealed that the OTX2 SAGE tags were most prevalent and showed

highest expression in medulloblastomas, whereas OTX2 tags were

absent or at low levels in other tumor types and in normal tissues. Tagnumbers of OTX2 were obtained normalized as tags of OTX2 per

200,000 total tags.

OTX2 as a Medulloblastoma Oncogene




Table 2. OTX2 expression in medulloblastomas

Sample

ID

Diagnosis* Fold of

OTX2

expressionby Q-PCR

c

OTX2 SAGE

tag numbers

per 200,000tags

b

Normal cerebellum 1.00 NA

Medulloblastoma cell lines

D487 NA 128.00 NA

D721 NA 90.62 NAD425 NA 72.17 NA

D283 NA 56.76 54

D384 Anaplastic,large cell

42.83 NA

D556 Anaplastic,

large cell

35.59 NA

D458 NA 35.03 NAD341 NA 13.10 72

D581 NA 0.91 NA

D324 NA 0.10 NA

MHH1 NA 0.01 0MCD-1 NA 0.01 NA

UW228 NA 0.00 NA

Medulloblastoma primary tumors

TB1244 Anaplastic,large cell

NA 77

TB285 Anaplastic NA 45

TB1273 Anaplastic NA 31

TB476 Anaplastic,large cell

328.43 NA

TB2235 Classic,

with focal

anaplasia

124.68 NA

TB2227 Anaplastic 100.50 NA

TB2223 Desmoplastic,nodular

85.36 NA

TB2226 Anaplastic 57.57 NA

TB1377 Anaplastic,

large cell

54.05 NA

TB1961 Anaplastic,

large cell

53.68 NA

TB830 Classic 44.09 NA

TB54 Classic,with focal

anaplasia

37.93 22

TB1339 Anaplastic,

large cell

36.49 9

TB2178 Desmoplastic,

with focal

anaplasia

18.10 NA

TB771 Anaplastic,

large cell

3.19 0


nodular

1.52 NA


nodular

0.91 NA


nodular

0.31 NA

TB2025 Desmoplastic 0.18 NA

(Continued)

Table 2. OTX2 expression in medulloblastomas (Cont’d)

Sample

ID

Diagnosis* Fold of

OTX2

expressionby Q-PCR

c

OTX2 SAGE

tag numbers

per 200,000tags

b

TB876 Desmoplastic,

nodular

0.17 0

TB100 Classic 0.05 0

TB2228 Classic 0.02 NATB1423 Desmoplastic 0.00 NA

Abbreviation: NA, data not available.*Histopathologic categories were defined by two board-certified

neuropathologist (REM, TJP) at Duke as follows. Desmoplasia:

connective tissue and/or pericellular reticulin fibers distributedthroughout the tumor. Large cell: cells with large open nuclei with

or without enlarged nucleoli. Anaplasia: high mitotic activity, nuclear

crowding, extensive apoptosis and necrosis, or extensive large cell

change. Classic pattern: low to moderate mitotic activity, apoptoticactivity, and necrotic foci.cFold of OTX2 expression was defined as the ratio of OTX2 expression

in tumor relative to normal.bThe SAGE data were obtained from the National Center forBiotechnology Information Cancer Genome Anatomy Project repos-

itory and the OTX2 SAGE tag, and its presence in the tumors was

identified by using the SAGE Anatomic Viewer.

Figure 2. OTX2 siRNAs repressed OTX2 expression and inhibited cell growthin medulloblastoma cell lines with OTX2 expression. A, inhibition of OTX2mRNA expression by OTX2 siRNAs. D283MED and D425MED, twoOTX2 -expressing medulloblastoma. Cell lines, were treated with OTX2 siRNA#1or siRNA#2. After 48 hours, cells were collected and mRNA was isolated forQ-PCR quantification of the level of OTX2 expression. B, OTX2 siRNAsinhibited medulloblastoma cell proliferation. Cell lines D283MED, D425MED,and D581MED# were treated with the OTX2 siRNA#1 or siRNA#2. After threedoubling times, the number of viable cells was determined by the MTT assay.D581MED# was an OTX2 -nonexpressing medulloblastoma cell line.

Cancer Research




tissues (Table 1). Consistent with SAGE analysis, quantification ofOTX2 mRNA by Q-PCR showed minimal expression in cerebellum,no detectable expression in other normal tissues tested, and noexpression in 18 glioblastoma multiforme tumors, the other majorbrain tumor type (data not shown). By contrast, high-level OTX2expression was identified in 21 of 33 (63%) medulloblastomasamples tested (Table 2).Medulloblastoma patients have widely disparate and often

unpredictable clinical outcomes. Two subtypes of medulloblastoma,large cell and nodular/differentiated, have documented worse andbetter prognoses, respectively (1). A recent Pediatric OncologyGroup study of 330 childhood medulloblastomas identified tumoranaplasia, found in 24% of tumors in their group, to be a marker ofaggressive clinical behavior (15). Among the 25 medulloblastomacases evaluated for OTX2 expression by Q-PCR or SAGE, 14 of 15(93%) cases with high OTX2 expression were anaplastic, whereasonly 2 of 10 (20%) cases with no OTX2 expression were classified asanaplastic (Table 2).To further examine the role of OTX2 expression, we disrupted

OTX2 expression in medulloblastoma cells using two different

OTX2 siRNAs against OTX2 . Repression of OTX2 expression bysiRNAs inhibited cell growth in the OTX2-expressing cell linesD283MED and D425MED, whereas there was no effect using thesame treatment on the OTX2-nonexpressing cell line D581MED(Fig. 2). We also identified a pharmacologic agent for manipulationof OTX2 expression in medulloblastoma cells. Exogenous retinoidscomprise a chemically related group of nuclear-acting lipid-solublehormones, which when exogenously applied have been shown todisplace or repress OTX2 expression in the embryonic nervoussystem and also in embryonal carcinoma cells through cis-actingelements of the OTX2 promoter (16, 17). We therefore examinedthe capacity of a strong retinoid, ATRA, for its effects on cellproliferation and apoptosis on OTX2-expressing and nonexpressingcell lines. Remarkably, ATRA abrogated cell proliferation in each ofthe seven OTX2-expressing cell lines in a dose-dependent mannerbut had no growth-inhibitory effects on the four medulloblastomacell lines with absent or minimal OTX2 expression (Fig. 3A).Furthermore, 2.0 AM ATRA markedly suppressed anchorage-independent tumor cell growth in each of the seven medulloblas-toma lines with OTX2 expression (Fig. 3B), whereas there was no

Figure 3. ATRA repressed OTX2 expression and inhibited cell growth in medulloblastoma cell lines with OTX2 expression. A, ATRA inhibited medulloblastoma cellproliferation. Medulloblastoma cell lines with OTX2 expression at high levels (solid symbol ) and lines without OTX2 expression (open symbol ) were treated with theindicated doses (0, 0.05, 0.5, and 2 Amol/L) of ATRA. After three doubling times, the number of viable cells was determined by the MTT assay. B, ATRA abrogatedanchorage-independent growth of medulloblastoma cells in soft agar. The indicated medulloblastoma cell lines were incubated with or without 2 Amol/L of ATRA.D283MED, D384MED, D425MED, D458MED, D487MED, D556MED, and D721MED expressed OTX2 at high levels, whereas D581MED expressed minimal OTX2 .C, inhibition of OTX2 mRNA expression by ATRA. D283MED, an OTX2 -expressing medulloblastoma cell line, was treated with the indicated ATRA doses (0, 0.5, 2, 5,and 10 Amol/L). After 6, 12, 24, and 48 hours, cells were collected and mRNA was isolated for Q-PCR quantification of the level of OTX2 expression. D, ATRA inducesmedulloblastoma cell apoptosis. D283MED, D425MED, D581MED, and MCD1 were treated with the indicated dose of ATRA (2 and 10 Amol/L) or vehicle control (DMSO).After 2 days, the cytoplasm of the cells was extracted and the histone-associated DNA fragments (nucleosome) enriched in the cytoplasm were quantified by theCell Death Detection ELISAplus method (Roche Diagnostic). D283MED and D425MED were OTX2 -expressing medulloblastoma cell lines, whereas D581 and MCD1were OTX2 -nonexpressing medulloblastoma cell lines. *, P < 0.05. #, OTX2 -nonexpressing cells.

OTX2 as a Medulloblastoma Oncogene




inhibitory effect on D581MED, which expressed OTX2 at a minimallevel (Fig. 3B). To illustrate that the growth inhibition was mediatedthrough the repression of OTX2 expression, we examined theexpression of OTX2 after ATRA treatment and found that ATRAblocked expression of OTX2 in a dose-dependent manner (Fig. 3C).We also showed that the ATRA-induced decrease in cell viability wasdue to increased cell apoptosis (Fig. 3D).In summary, this study shows the power of digital karyotying

as a high-resolution whole-genome approach for the isolation ofindividual oncogenes. Through analyses of genomes and tran-scriptomes, we identified OTX2 as being overexpressed in themajority of medulloblastomas with anaplastic histopathologicfeatures and as being genetically amplified in a subset of them.The genetic alterations of OTX2 provide cogent evidence fora pathogenic role, as tumor expression data alone cannot beinterpreted with respect to cause and effect (18). Further experi-ments are necessary to investigate the possibility that abnormalOTX2 expression may result in the dysregulation of the develop-mental programs in cerebellar progenitor cells and cause neo-plastic transformation. It is of interest that other developmentalregulatory pathways have been shown to be altered by expressionor mutation in medulloblastomas. In particular, the hedgehog/patched signaling pathway is required for the proliferation andmaintenance of cerebellar progenitor cells, and mutations of thecomponents of this pathway have been identified in medulloblas-tomas. It will be interesting to determine if OTX2 is part of thissignaling network or if it belongs to a parallel/alternate pathway.Finally, the prospect for the rational use of a new therapeutic

agent against medulloblastoma should be discussed. This studyshowed that ATRA repressed OTX2-expression and inhibited

OTX2-expressing medulloblastoma cell growth. Although ATRAand other retinoids may affect multiple molecular pathways, theconnection between OTX2 repression and growth inhibition effectof ATRA suggests that OTX2 expressing medulloblastomas may beamenable to therapy with retinoids. Previous studies have shownthat pharmalogically relevant doses of ATRA induce apoptosis inmedulloblastoma cells, although a connection with anaplastichistology, or OTX2 expression was not established (19, 20). ATRA isapproved clinically for the treatment of propmyelocytic leukemia, adisease in which another nuclear receptor is a predominantdeterminant of pathogenesis (21). Our studies of ATRA inmedulloblastoma, in conjunction with the studies of others, laythe conceptual framework for clinical trials of retinoids in thetreatment of a commonly lethal pediatric brain tumor.

Acknowledgments

Received 9/9/2004; revised 11/1/2004; accepted 11/18/2004.Grant support: Pediatric Brain Tumor Foundation Institute at Duke, Duke

Comprehensive Cancer Center support grant 2P30CA14236; NIH grants NS20023-21and R37CA11898-34; Brain Tumor Specialized Programs of Research Excellence5P20CA096890-02; American Brain Tumor Association; Neurosurgery ResearchEducation Foundation; Accelerate Brain Tumor Cure Foundation; National CancerCenter; and National Cancer Institute Cancer Genome Anatomy Project.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Bert Vogelstein for pre-reviewing the article and providingcritical comments; Drs. Henry Friedman, Allan Friedman, Timothy George,Sridharan Gururangan, and B.K. Ahmed Rasheed, David Lister, David Stitzel,Nancy Bullock, Linda Cleveland, Diane Satterfield, Lisa Ehinger, and Stephen Keir inthe Brain Tumor Center at Duke for their support and assistance; and Dr. William Freedat NINDS, Bethesda, MD and Dr. John Silber at the University of Washington, Seattle,WA for the medulloblastoma cell lines MCD-1 and UW-228, respectively.

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2005;65:919-924. Cancer Res Chunhui Di, Shaoxi Liao, David C. Adamson, et al.

Retinoic AcidTransWhose Product can be Targeted by All- as a Medulloblastoma OncogeneOTX2Identification of

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