GENETIC STUDIES OF GENETIC STUDIES OF C-MYCC-MYC ONCONGENE ONCONGENE AND AND ROR1ROR1 GENE OVEREXPRESSION IN GENE OVEREXPRESSION IN

EPENDYMOMASEPENDYMOMAS

YUEMIN DINGYUEMIN DING

Neuro-oncology GroupNeuro-oncology Group

Department of Molecular NeuroscienceDepartment of Molecular Neuroscience

Institute of NeurologyInstitute of Neurology

Queen Square, LondonQueen Square, London

Supervisor: Dr Tracy WarrSupervisor: Dr Tracy Warr

BACKGROUNDBACKGROUND

• EpendymomasEpendymomas are glial tumors that aare glial tumors that arise from the ependymal lining of the rise from the ependymal lining of the ventricular system of the CNS. ventricular system of the CNS.

• They represent the third most frequent They represent the third most frequent brain tumor in children. brain tumor in children. (Hamilton RL (Hamilton RL et alet al. 1997). 1997)

• The genetic events that contribute to tThe genetic events that contribute to the pathogenesis of paediatric ependymhe pathogenesis of paediatric ependymoma are poorly defined. oma are poorly defined. (Ward S (Ward S et alet al. . 2001)2001)

BACKGROUNDBACKGROUND

• The expression of >12,000 genes in a set of 11 ependymoThe expression of >12,000 genes in a set of 11 ependymoma samples were determined using oligonucleotide micrarma samples were determined using oligonucleotide micrarrays analysis. Oncogene rays analysis. Oncogene c-mycc-myc and gene and gene ROR1ROR1 were identi were identified to be highly expressed in the tumour samples. fied to be highly expressed in the tumour samples.

• Previous experiments failed to show extra copies of c- myc gene in ependymomas by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH).

Previous finding of our groupPrevious finding of our group

BACKGROUNDBACKGROUND

c-myc oncogene

c-Myc

CHR # 8

Cell growth

proliferation differentiation

apoptosisCellular processes

BACKGROUNDBACKGROUND

c-Myc c-Myc c-Myc

c-Myc c-Myc c-Myc

c-Myc c-M

yc

c-Myc c-M

yc

!— c-myc has emerged as a central oncogenic switch in many human cancers. (Stella Pelengaris 2002)

BACKGROUND BACKGROUND

MechanismsMechanisms lead to gene overexpression lead to gene overexpression

• Chromosome duplication Chromosome duplication

• Gene amplificationGene amplification

• Point mutationPoint mutation

• Regulators dysfunctionRegulators dysfunction

overexpression of normal product

chromosome duplication

gene amplification

point mutation

A - T C - G G - CA - TT - A

A - T C - G G - CG - CT - A

overexpression of normal product

Loss of function mutation

BACKGROUNDBACKGROUND

• The expression of >12,000 genes in a set of 11 ependyThe expression of >12,000 genes in a set of 11 ependymoma samples were determined using oligonucleotide moma samples were determined using oligonucleotide micrarrays analysis. Oncogene micrarrays analysis. Oncogene c-mycc-myc and gene and gene ROR1ROR1 were identified to be highly expressed in the tumour sawere identified to be highly expressed in the tumour samplesmples. .

• Previous experiments failed to show extra copies of c- myc gene in ependymomas by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH).

Previous finding of our groupPrevious finding of our group

HYPOTHESISHYPOTHESIS

The regulator upstream in The regulator upstream in the c-Myc pathway was abthe c-Myc pathway was abnormal or dysfunctional, enormal or dysfunctional, eg. loss of function mutatiog. loss of function mutation in MM-1.n in MM-1.

BACKGROUNDBACKGROUND

Receptor tyrosine kinase-like orphan receptor 1 (ROR1)

FRZ: frizzled module

Wnt pathway

Oncogene WNT5A

HYPOTHESISHYPOTHESIS

ROR1 plays a role in the pathogenesis of ependymomas partially mediated through Wnt-ROR pathway.

AIMSAIMS

• To investigate the mutation status of the To investigate the mutation status of the MM-1MM-1 gene, gene, which may play a role in the development of ependywhich may play a role in the development of ependymomas by deregulating c-momas by deregulating c-mycmyc expression. expression.

• To identify the association between the genetic changTo identify the association between the genetic changes of the es of the ROR1ROR1 gene and the pathogenesis of ependy gene and the pathogenesis of ependymomas via Wnt pathway.momas via Wnt pathway.

MATERIALS AND METHODSMATERIALS AND METHODS

• Tumour specimensTumour specimens

• PCR/RT-PCRPCR/RT-PCR

• SSCP analysisSSCP analysis

• DNA sequencingDNA sequencing

IN1 Age

2 Sex

3 Grade

4 P/R

5 Location Survival

6

(months)

Gains7 Losses

7

772 4 M SE P Supratentorial 72(D) 4 1p 16p

1134 2 F E P Posterior fossa 17(D)

1231 7 F E P Supratentorial 64(D)

1258 15 M E R Posterior fossa 98(A) 6q 17p

18q 22

1497 1.25 M E R Posterior fossa 45(D) 6q

2186 12.5 F E P Posterior fossa 118(A)

2443 1.8 F AE P Posterior fossa 52(A)

2682 0.66 F E P Posterior fossa 11(D)

2941 2.58 M E P Posterior fossa 24(A)

3008 10.5 M E P Posterior fossa 27(A)

3014 6.75 M E P Supratentorial 23(A) 4q 5q

6q 13q

1p 9q 12q

16p 17q

19q 22

3108 4 M AE R Posterior fossa 21(A)

Ependymoma case information (sourse: cell culture)

Key to table:

IN1: Institute of Neurology assigned number

Age2: Age in years at diagnosis

Sex3: M = male; F = female

Grade4: SE = subependymoma; E = ependymoma; AE = Anaplastic ependymoma

P/R5: P = primary sample; R = recurrent sample

Survival6: survival in months from date of diagnosis

Gains7 and losses7: detected by CGH

MATERIALS AND METHODSMATERIALS AND METHODS

• Tumour specimensTumour specimens

• PCR/RT-PCRPCR/RT-PCR

• SSCP analysisSSCP analysis

• DNA sequencing DNA sequencing

MATERIALS AND METHODSMATERIALS AND METHODS

• Tumour specimensTumour specimens

• PCR/RT-PCRPCR/RT-PCR

• SSCP analysisSSCP analysis

• DNA sequencingDNA sequencing

MATERIALS AND METHODSMATERIALS AND METHODS

• Tumour specimensTumour specimens

• PCR/RT-PCRPCR/RT-PCR

• SSCP analysisSSCP analysis

• DNA sequencingDNA sequencing

1 2

34

ProtocolProtocol

Extract DNA from frozen cell cultures of ependymoma samples

Amplify target gene by PCR technique

Search the Genomic Database and design the primers

Detect mutations by SSCP analysis

Direct DNA sequencing

RESULTSRESULTS

MM-1

Expression level of c-Myc

SSCP analysis of MM-1

DNA sequencing of MM-1Fig 1 RT-PCR studies of c-myc. cDNAs were synthesized from RNA of the tumour samples and the normal brain (CC). β-actin was used as the internal control. DNA marker used is the 100bp ladder. c-myc was overexpressed in tumour samples compared with the CC.

A

B

Fig. 2 Representative results of SSCP analysis of MM-1. Lanes 1–10, samples of ependymomas. A, MM-1 exon 4-5; B, MM-1 exon 6. Arrowhead highlights abnormal migrating SSCP bands.

RESULTSRESULTS

ROR1ROR1

Expression level of Expression level of ROR1ROR1

SSCP analysis of SSCP analysis of ROR1ROR1

DNA sequencing of DNA sequencing of ROR1ROR1

RESULTSRESULTS

Fig. 3 RT-PCR analysis of ROR1. cDNAs were synthesized from RNA of the tumour samples and the normal brain (CC). β-actin was used as the internal control. DNA marker used is the 100bp ladder. Note ROR1 was overexpressed in tumour samples compared with CC.

Fig. 4 Representative results of SSCP analysis of ROR1. Lanes 1–11, samples of ependymomas. A, ROR1 exon 7; B, ROR1 exon 8. Arrowhead highlights abnormal migrating SSCP bands.

A

B

3008 For

3008 Rev

2941 For

2941 Rev

A B

Fig. 5 Sequence confirmation of mutations identified by SSCP analysis (ROR1 gene). Black arrows indicate the heterozygous C → T mutation in exon 7 of the ependymoma samples, which were confirmed in both sequencing reactions. (A, sample IN3008; B, sample IN2941)

1258 For

1258 Rev 2443 Rev

2443 For2682 For

2682 Rev

Fig. 6 Sequence confirmation of mutations identified by SSCP analysis (ROR1 gene). Black arrows indicate the heterozygous (A and B) or homozygous (C) A → G mutation in exon 8 of the ependymoma samples, which were confirmed in both sequencing reactions. (A, sample IN1258; B, sample IN2443; C, IN2682)

A B C

RESULTSRESULTS

Sample Exon Nucleotide Change

Codon Amino Acid change

1134 No mutation 1231 No mutation 1258 8 GTA to GTG 451 Silent mutation 1497 No mutation 2186 No mutation 2443 8 GTA to GTG 451 Silent mutation 2682 8 GTA to GTG 451 Silent mutation 2941 7 ACC to ACT 348 Silent mutation 3008 7 ACC to ACT 348 Silent mutation 3014 No mutation 3108 No mutation

Table 1 Sequence changes in the ROR1 gene

Immunoglobulin domain (72 – 133) FRZ domain (160 – 297)Kringle domain (313 – 391) Phabdovirus spike glycoprotein (411 – 457) Protein kinase domain (473 – 746)

Fig. 4.2 The structures of ROR1 and SNP location. The silent mutation on codon 348 was located at Kringle domain and the other mutation on codon 451 was at Phabdovirus spike glycoprotein.

NM_005012.1 [1p32_p31] 937 aa

348:T→T

451:V→V

CONCLUSIONCONCLUSION

• We failed to detect mutations in MM-1 in ependymomas by DNA sequencing. But it didn’t preclude the possibility that mutations in MM-1 abolished its suppressor function, which lead to c-Myc overexpression, because loss of function mutations may have been missed in the present sequencing analysis.

• Two silent mutations were found in ROR1 but they present little relevance in the Wnt pathway in ependymomas. Association of these mutations in ROR1 to cancer risk require further studies.