COMPREHENSIVE ANALYSIS OF DNA COPY NUMBER VARIATIONS AND GENE EXPRESSION IN OSTEOSARCOMA
Nalan Gokgoz, Atta Goudarzi, Cheryl Wolting Jay S. Wunder and Irene L. Andrulis
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital Toronto, ON, Canada.
Connective Tissue Oncology Society Meeting November 1, 2013
High resolution approaches to identify genes and pathways predictive of outcome in OS Gene expression profiling by Microarray Analysis
Identification of the most relevant biological pathways for list of discriminative genes by Ingenuity Pathway Analysis
Identification of the significant effectors and organizing networks in OS metastasis by Dynamo (Taylor and Chuang)
Interrogation of biological pathways and networks
Investigation of Copy Number Changes by Illumina SNP array technology
Detection and characterization of alterations
Analysis and visualization by Genome Studio and GAP
Identification of significant recurrent targets by GISTIC
Identification and Characterization of Molecular Alterations in Osteosarcoma
High-grade Intramedullary 63 patients
No Metastasis at Diagnosis46 patients
Metastasis at Diagnosis17 patients
No Metastasis 4 years post Dx. (29 patients)
Metastasis within 4 years Dx.(17 patients)
A B
A1
A2
PATIENT COHORT
No Metastases 4 years post Dx (A1)vs
Metastases within 4 years Dx (A2)
18981 cDNAs
T-statistic
p<0.001
(BrB Array Tools)
n=53 genes
for tumor classification/clustering
Statistical validation by Leave-One Out cross-validation method
Molecular validation by Real-Time Analysis
Outcome of the Patients Presenting with “no Metastases”
No Mets.
4 yrs post Dx. Mets. within
4 yrs post Dx.
MICROARRAY ANALYSIS
Gene AGene C
.
.Gene XGene Y
Interactions and Relationships between molecules in set
Networks
Pathways with which molecules in set are associated
Pathways
Functions with which molecules in set are associated
Functions
Upstream regulators that may be responsible for observed increase/decrease in expression
Upstream Regulators
Molecule Set
Ingenuity Pathway Analysis
Summary of IPA in OS metastasis
Networks
cell morphology, organization, hematopoiesis
Pathways
Rac/Rho, actin cytoskeleton
Functions
hematopoiesis, cell movement
Regulators
Fas, Fos, SP1, SREBF1
Signaling by Rho Family GTPases
Lower expression in A2Higher expression in A2
A1 – No metsA2 – Mets in 4 yrs
GENETIC NETWORKS in OS METASTASIS
• The PRKCε, RASGPR3 and GNB2 networks differentially activated• DLG2 network differentially organized• The PRKCε, RASGPR3 and GNB2 networks are potential effectors of DLG2
Significant Networks• Transport • Translation • Signaling
Protein Kinase C Epsilon and Genetic Networks in Osteosarcoma Metastasis; A Goudarzi, N Gokgoz, M Gill, D Pinnaduwage, D. Merico, J.S Wunder and IL Andrulis, Cancer, 2013, 5, 372-403
Osteosarcoma and Copy Number Alterations
Illumina 610-Quad Whole-genome genotyping beadchip
Coverage includes >14,000 CNV regions and 550K evenly spaced TagSNPs from HapMap data
High Resolution: Spacing 2.7 kb Includes markers in the unSPNable Genome Allows detection of SNPs, Copy Number
Variation and Genotype Reference Genotype: Canonical genotype
clusters (200 HapMap DNA genotype data) 44 Osteosarcoma Tumor DNA
Validation by Real Time PCR
25 of them with matched blood DNA
Complexity of OS Tumour Genome (Analysis by Genome Studio)
Blood DNA
Tumour DNA
Allele Frequency
BB AB AA
LogR Ratio
COL12A1
COL9A1
AF086303
CDK4
MDM2
COL4A1
COL4A2
LIG4
MYR8
COPS3
NCORI
PMP22PPFIBP1FGFR1OP2
Recurrent Copy Number Gains in OS identified by GISTIC (Genome Identification of Significant Targets in Cancer)
**
*
*Same family genes
q v
alu
e
Recurrent Copy Number Losses in OS identified by GISTIC
LOC285194
CNTNAP2
CDKN2A
MTAP
DLG
2
RB1
TP53
GRIK2
**
*
DOCK5
*Same family genes
q v
alu
e
NAALADL2
One of the most disorganized genetic networks in metastatic OS tumours.
The PRKCε, RASGPR3 and GNB2 networks are potential effectors of DLG2
Tumour suppressor function of dlg2 in Drosophila
Scribble complex (SCRIB, DLG1-4 and LGL1/2) deregulation in Prostate Cancer
DLG2 implicated in Wilms Tumour
Implication of DLG2 as a tumour suppressor in cancer
DLG2 (discs, large homolog 2) Channel associated protein of synapse 110 Chromosome 11q14
Member of the membrane-associated guanylate kinase (MAGUK) family.
• PDZ domains; interaction with signalling proteins at postsynaptic sites • SH3 domains are found in proteins of signaling pathways regulating the
cytoskeleton and regulate the activity state of adaptor proteins and other tyrosine kinases
• GuKinase Domain; catalyzes ATP-dependent phosphorylation of GMP to GDP
Gene: 2 MB, 33 alternative spliced transcripts Longest transcript :3.7KB, 26 Exons• Expression site: Brain, hypothalamus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20U2OS
SaOS
SaOs-L
M7LM
70.000
0.050
0.100
0.150
0.200
0.250
0.300
OS Tumours
DLG
2/ST
AM
2 Relative Expression of DLG2 in OS tumours and cell-lines
Deletion of DLG2 gene detected by SNP array
SiRNA Knockdown of the DLG2 Gene in U2OS 70% knockdown at 72 hours
48 72 960
20
40
60
80
100
120
140
Work in Progress
The effect of DLG2 knockdown in Cell viability and growth by XTT assay Migration by scratch assay
Sequencing of DLG2 gene for inactivating mutations
We identified a 53-gene expression signature that may predict outcome of OS patients with localized tumours.
High-resolution approaches identified candidate pathways and networks that may be biologically relevant in OS.
Cell morphology and organization pathways may be involved in OS metastasis.
A large number of chromosomal aberrations were detected in OS tumours by SNP array technology.
The DLG2 gene that is deleted in 20 percent of the OS cases and belonging to a significantly disorganized metastatic OS network and was chosen for further functional analysis.
Further experiments will be performed to investigate the functional role of DLG2 in cell growth, proliferation and migration.
CONCLUSION
Acknowledgement
Mount Sinai HospitalOrthopedic Surgeons
Hospital for Sick Children D.Malkin
Vancouver General Hospital C.Beauchamp
R. Kandel
University of Washington E.Conrad III
Royal Orthopedic Hospital R.Grimer
Memorial Sloan-Kettering J.Healey
Mayo Clinic M.Rock/ L.Wold
Andrulis and Wunder Lab
S. Bull
R. ParkesI. Andrulis
J. Wunder
Andrew Seto
During progression from tumour growth to metastasis, specific integrin signals enable cancer cells to detach from neighbouring cells, re-orientate their polarity during migration, and survive and proliferate in foreign microenvironments. There is increasing evidence that certain integrins associate with receptor tyrosine kinases (RTKs) to activate signalling pathways that are necessary for tumour invasion and metastasis. The effect of these integrins might be especially important in cancer cells that have activating mutations, or amplifications, of the genes that encode these RTKs.