alternative splicing as driving force in cancer · 2015. 12. 4. · transcript3 calculate psi...

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