Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies

Maxwell Lee

National Cancer InstituteCenter for Cancer Research

Laboratory of Population Genetics

September 29th, 2010

Three Parts

• Genomic analysis of breast cancer and functional studies of novel oncogenes.

• Large-scale analysis of allele-specific gene expression and epigenetic modifications.

• Deep-sequencing analysis of breast cancer transcriptome.

Our Approach to Understanding the Etiology of Breast Cancer

breast tumors

genomics

cancer-related genes

epigenomics

Part 1

Mits Kadota

Clinical Information of Our Primary Breast Tumors (CHTN)

A

Numbers refer to tumor counts in each category

Part 1

Primary Breast Tumors:DNA Copy Number Variation

161 tumors

Part 1

chro

mos

ome

161 breast tumors

putative novel oncogenes

Affymetrix SNP5 array

1q

8q

traditional approach

our approach

size of focal amplification

multiple genes 1 gene

frequency of tumors with

amplificationcommon

Don’t require common but requiremultiple occurrence

Focal Amplification of TBL1XR1 in Breast Tumors

Part 1

Focal Amplification Detected in Primary Breast Tumors

Part 1

Gene Amplification and Up-regulation at RNA and Protein Levels of TBL1XR1 in Breast Tumors

focal amplification

Hypothesis:

additional mechanisms for up-regulation

amplification orup-regulation

DNA

RNA protein

Part 1

genomic alteration

RNAover-expression

proteinover-expression

Primary Breast Tumors:Frequent Over-expression of TBL1XR1

Well-diff poorly-diff

negative 16 4

positive 35 15

negative31%

positive69%

N=84odds ratio = 1.7 N=70

TBL1XR1 staining increases in poorly-differentiated tumors

Part 1

In collaboration with Dr. Junya Fukuoka

24 hour

cell migration (scratch assay)

0 hour

control

TBL1XR1- shRNA

TBL1XR1-shRNA Knockdown of MCF10CA1h Cells Suppresses In Vitro Cell Migration

Western Blot

Part 1

MCF10A

HRASMCF10AT MCF10CA1h

MCF10CA1a

In collaboration with Dr. Lalage Wakefield

TBL1XR1-shRNA Knockdown Suppresses In Vivo Tumor Growth

MCF10CA1h Control-shRNA TBL1XR1-shRNA

Tum

or v

olum

e (m

m3 )

Day 39

N=10 N=10 N=14

N=5 N=5 N=7mice

implants

Part 1

Kadota et al. Cancer Research. 2009

Two Complementary Approaches

Part 1

SNP array 10 kb resolution

hundreds of tumors

Functional studies

Next-generation sequencingsingle nucleotide resolution

a few samples

Functional studies

3 Major Goals of Deep Sequencing Analysis of Breast Cancer Transcriptome

Part 2

1) To identify genes with differential expression between tumor and normal tissues.

2) To identify somatic mutations in breast tumors.

3) To characterize allele-specific gene expression in tumor and normal tissues.

Histopathological Data of Tumors

sampletumor

contentER PR HER2 stage grade invasion node type

BT1 100 - - + T3N3 3 yes positive invasive ductal carcinoma

BT2 100 - - - T2N3c 3 yes positive invasive ductal carcinoma

BT3 100 - - + T3 3 yes invasive ductal carcinoma

BT4 95 - - - T2N0 3 yes negative Invasive ductal carcinoma

Part 2

Summary of Sequence Reads and BWA Mapping

76 base and 108 base pair-end sequences

mapping pipeline

Assemble bam files

hg18 reference sequencemRNA refseqESTcombination of any two exons

Part 2

Mil

lion

rea

ds

20

40

60

140

120

100

80

splicing junction

The Number of Genes that Showed Differential Expression between Tumor and Normal Tissues

tumor number of genes that were down-regulated in tumor (FDR=0.1)

number of genes that were up-regulated in tumor (FDR=0.1)

BT1 14602 3644

BT2 13840 2905

BT3 13327 7652

BT4 12500 7584

Part 2

SOX10 Is Down-regulated in a Tumorchr22:36686044-36722706

BN1

BN4

BN3

BN2

BT1

BT4

BT3

BT2

normal

tumor

Part 2

MMP9 Is Up-regulated in Tumorschr20:44062819-44086029

triple negative

BN1

BN4

BN3

BN2

BT1

BT4

BT3

BT2

triple negative

Part 2

Comparison of Up-regulated Genes between Tumors

BT1 BT2 BT3 BT4

BT1 100 5 5 12

BT2 5 100 10 29

BT3 5 10 100 8

BT4 12 29 8 100

triple negative

triple negative

Part 2

2 triple negative tumors

HER positive

HER positive

An isoform of GNAS Is Down-regulated in Tumors

matpat

bi-allelic

BN1

BN4

BN3

BN2

BT1

BT4

BT3

BT2

Part 2

Somatic Mutation Summary

BT1 BT2

BT3 BT4

tumor mutant reads > 10normal mutant read = 0tumor mutant fraction > 0.1normal mutant fraction < 0.05normal reference reads > 10

Part 2

Validation of Somatic Mutations in Genomic DNA

tumor

normal

P952R

ESYT1

tumor

normal

K217Q

RYBP

Part 2

Summary of Validation Experiments

Part 2

gene tumor mutation mutant fraction description

ESYT1 BT4 P952R 1 extended synaptotagmin-like protein 1

G3BP2 BT2 S48T 0.2GTPase activating protein (SH3 domain) binding

protein 2; oncogene, sequesting TP53

GPRC5A BT2 S59C 0.4G protein-coupled receptor, family C, group 5,

member A; tumor suppressor in lung cancer

INO80B BT1 P306L 0.2INO80 complex subunit B; tumor suppressor in

prostate cancer

OSTF1 BT3 L20P 0.2 osteoclast stimulating factor 1

PIK3CA BT3 G1049R 0.1 phosphoinositide-3-kinase, catalytic, alpha polypeptide

RAPH1 BT2 D1199N 0.5Ras association and pleckstrin homology domains

1 isoform 1; in JHU screen

RYBP BT2 K217Q 0.5RING1 and YY1 binding protein; tumor

suppressor, stabilizing TP53

SHQ1 BT4 K193M 0.5 protein SHQ1 homolog

USP6NL BT3 V536I 0.3 USP6 N-terminal like

VIM BT2 E153A 0.2 vimentin

Red highlights deleterious change by SIFT

Allelic Variation in Gene Expression is Common in the Human Genome

cDNA

Affymetrix HuSNP array

Lo et al. Genome Res. 2003

normal human fetal tissues

Genomic imprinting

50~100 genesall or noneparental origin

Allelic gene expression

20%~50% genesquantitative differencesequence, cellular context, etc.

> 2-fold difference

277 genes

326 genes

Part 3

What Determine Allelic Variation in Gene Expression?

• Epigenetic mechanism

• Genetic mechanism

Part 3

Allele-specific ChIP-on-chip Studies in Lymphoblastoid Cell Lines from CEPH Families

Part 3

Allele-specific ChIP-on-chip Studies in Lymphoblastoid Cell Lines from CEPH Families

1347 1362

relative allelic signal (RAS)

allele A/(allele A + allele B)

0 0.25 0.5 0.75 1 RAS

activechromatin

inactivechromatin

Pat Mat

A B

DNA or

LIT1, an imprinted gene

Part 3

RAS

A /(A+B)

Clustering of Samples Based on Family Using Allele-specific Chromatin Features

Part 3

Inheritance Analysis of Allelic Histone H3 Acetylation at the TMEM16D Locus

Informative for SNPinterrogated on SNP array (rs938335)

RASA/ (A + B)

L: low H3AcM: medium H3AcH: high H3Ac

L H HH

MM

Part 3

Kadota et al. PLoS Genet. 2007

Clustering of 42 Primary Breast Tumors by the Degree of Mono-allelic Methylation in Chromosome Arms

A

early stagelate stage late stagetriple neg.

Part 3

Acknowledgments

Mitsutaka KadotaHoward YangBeverly DuncanSheryl Gere

Misako SatoAkira OoshimaLalage Wakefield

Robert CliffordRichard FinneyShuang CaiChunhua YanMichael EdmonsonDaoud MeerzamanKen Buetow

Nan HuChaoyu WangHua SuPhil Taylor

Shun-Ichiro Kageyama Takuya NagataJunya Fukuoka Kazuhiro Tsukada

NCI/CCR

Kent Hunter

Alisa GoldsteinNCI/DCEG

Barbara DunnNCI/DCP

Japan/Toyama Univ.

Jiuping JiNCI/DCTD