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GENETIC STUDIES OF C-MYC ONCONGENE AND ROR1 GENE OVEREXPRESSION IN EPENDYMOMAS. YUEMIN DING Neuro-oncology Group Department of Molecular Neuroscience Institute of Neurology Queen Square, London Supervisor: Dr Tracy Warr. BACKGROUND. - PowerPoint PPT Presentation
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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.