alternative splicing as driving force in cancer · 2015. 12. 4. · transcript3 calculate psi...
TRANSCRIPT
Results
Calculate Splicing Change (PSI)
Normal samples Tumor samples
PS
I
PS
I
Transcript1
Transcript2
Transcript3
Calculate PSI
Skipping exon
Mutually exclusive exons
Alternative 5’ splice-site
Alternative 3’ splice-site
Retained intron
Alternative first exon
Alternative last exon
Define Alternative Splicing Event coordinates
MEME-Suite
Differentially Spliced events
Alternative Splicing as Driving Force in Cancer Endre Sebestyén, Babita Singh*, Belén Miñana, Amadís Pagès, Francesca Mateo, Miguel
A Pujana, Juan Valcarcel, Eduardo Eyras *Computational Genomics Group, University of Pompeu Fabra, PRBB, Barcelona (Spain)
HRAS
Anti-proliferating
Proliferating
Exon 4 Exon 5 IDX
FAS
Pro-apoptotic
Anti-apoptotic
Exon 5 Exon 7 Exon 6
Cell
Gets Old
Gets signal for apoptosis
Ignores apoptotic signal
Tumor cells evade growth inhibitory
signals and keep on growing
VEGFA
Anti-angiogenic
Pro-angiogenic
Exon 7 Exon 8
Stop
Stop
Tumor cells develop blood vessels to get
blood supply
hTERT
Exon 6 Exon 8 Exon 7 Exon 9
Non-functional telomerase
Functional telomerase
Telomers are found at the
end of chromosomes, they get shorter after
every cell division
For
unlimited replicative potential, cancer cell recruits telomerase
No more replication
No more telomere
Exon 10 Exon 12 Exon 11
Increased cell motility
MSTR1
Normal cell motility
Tumor cells leave the tissue of
origin, invade and metastasize to other parts of
body
Tumor cells evades
apoptotic signals and keep
on working
Cell + growth signal
Growth Inhibitory Signal to stop
division
DNA Samples
RNA Samples
Clinical Data
Sequencing and Data generation
Data Download
Motif Enrichment Analysis
Exon Exon
mRNA
YYYYYY A AG GU
Splicing Regulatory Sites
Inside a gene exons are
separated by introns
Removal of introns create mature RNAs, that then translates to protein
Different exon arrangements lead to different protein isoforms. This process
is known as Alternative Splicing (AS). Protein Isoforms
AS is regulated by network of RNA binding proteins (RBPs) & Splicing Factors (SFs)
DNA/RNA sample preparation and Aliquot ids
Differential Expression analysis of Gene s
Gene Expression data file (counts)
Data Cleaning
+ Data
Normalization
CGHub/TCGA-data portal
Head & Neck (HNSC) Thyroid (THCA) Breast (BRCA) Lung (LUSC) Lung (LUAD) Liver (LIHC) Kidney (KICH Kidney (KIRP) Kidney (KIRC) Bladder (BLCA) Colon (COAD) Prostrate (PRAD) Ovary (OV) Others..
Cancer Types
Samples collected from 11,000 patients, for 33 cancer types
Matched normal and tumor samples extracted from each patient
Reference: Sebestyén and Singh et al., Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks (doi: http://dx.doi.org/10.1101/023010)
The Cancer Genome Atlas (TCGA) provides a platform for researchers to analyze data sets generated by TCGA. We used these datasets to study
alternative splicing in multiple cancer types
Exon Exon E
TPM = Transcripts Per Million
RNA-Seq
Normal samples Tumor samples
Calculate transcript abundance of each gene
TPM1
TPM2
TPM3
gene
Differential Splicing Analysis
Breast (BRCA) RBFOX2, RBM17 RBM39, SRSF1, SRSF5, TRA2B, ELAVL1,HNRNPA1
Lung (LUSC,LUAD) TRA2B, HNRNPA1 RBM10, HNRNPA2B1 U2AF1, RBM17, RBM5, RBM6, RBM10, QKI
Kidney
(KICH,KIRC,KIRP) SRRM1, RBM8A, QKI, SAMD4A
Liver (LIHC) ESRP1,SAMD4A, RBMS1, RBMS3
Prostrate(PRAD) KHDRBS1, RBM17
Colon (COAD) SRSF1, HNRNPF, HNRNPK, RBM17, SRSF1, SRSF6, SF1
Head & Neck (HNSC) HNRNPK, PCBP2, RBM28, CELF2, CELF3, CELF6
Thyroid (THCA) SRSF1,RBFOX1,2,3, MBNL2,3, SAMD4A
RNA Binding Proteins Affecting
Differential Splicing in Cancer
Differential splicing of NUMA1 is controlled by MBNL1
protein
Differential Splicing of NUMA1 gene in Breast Cancer
NUMA1 exon 16
10 11 12 13 14
Scr1
10
0 n
M
20
nM
60
nM
siMBNL1 exon 3
10
0 n
M
20
nM
60
nM
10
0 n
M
siMBNL1 exon 5
20
nM
60
nM
10
0 n
M
siMBNL1 both
Scr
1 2
0 n
M
siQKI
e4
20
nM
e6 2
0n
M
Bo
th 2
0 n
M
1 2 3 4 5 6 7 8 9
PUM
normal tumor
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
NUMB exon 9 (LUAD)
type
PS
I
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PSI
Normal Tumor
(~900 mutational drivers from the literature)
Total Events found differentially spliced in cancer
Samples
Differential Events
Cancer drivers are enriched in differentially spliced events
Alternative Splicing of NUMA1 gene leads to
genome instability
R= -0.4
R= -0.33
Centriole counting
MBNL1
MBNL proteins are found to be frequently down regulated in
tumors and their motif is enriched in diff. spliced events
NUMA1 NDEL1 ITGA6 PALM VPS29
RAB11FIP3 EPB41L1
DST
NUMB SLK
EPB41 SMARCC2
EXOC1 CD47
MYO18A STX2 CD47
MARK2 GOLGA4
Events with MBNL1 motif
Events without MBNL1 motif
MBNL1 potentially affects the AS of many cancer
drivers
siRNA mediated MBNL1 depletion in normal breast cell-line induces exon skipping of NUMA1, recapitulating splicing pattern of tumor samples.
MCF10A