Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and...
-
Upload
eugene-young -
Category
Documents
-
view
217 -
download
0
Transcript of Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and...
![Page 1: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/1.jpg)
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
![Page 2: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/2.jpg)
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.
![Page 3: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/3.jpg)
Our Approach to Understanding the Etiology of Breast Cancer
breast tumors
genomics
cancer-related genes
epigenomics
Part 1
Mits Kadota
![Page 4: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/4.jpg)
Clinical Information of Our Primary Breast Tumors (CHTN)
A
Numbers refer to tumor counts in each category
Part 1
![Page 5: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/5.jpg)
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
![Page 6: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/6.jpg)
Focal Amplification of TBL1XR1 in Breast Tumors
Part 1
![Page 7: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/7.jpg)
Focal Amplification Detected in Primary Breast Tumors
Part 1
![Page 8: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/8.jpg)
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
![Page 9: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/9.jpg)
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
![Page 10: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/10.jpg)
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
![Page 11: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/11.jpg)
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
![Page 12: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/12.jpg)
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
![Page 13: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/13.jpg)
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.
![Page 14: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/14.jpg)
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
![Page 15: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/15.jpg)
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
![Page 16: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/16.jpg)
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
![Page 17: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/17.jpg)
SOX10 Is Down-regulated in a Tumorchr22:36686044-36722706
BN1
BN4
BN3
BN2
BT1
BT4
BT3
BT2
normal
tumor
Part 2
![Page 18: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/18.jpg)
MMP9 Is Up-regulated in Tumorschr20:44062819-44086029
triple negative
BN1
BN4
BN3
BN2
BT1
BT4
BT3
BT2
triple negative
Part 2
![Page 19: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/19.jpg)
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
![Page 20: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/20.jpg)
An isoform of GNAS Is Down-regulated in Tumors
matpat
bi-allelic
BN1
BN4
BN3
BN2
BT1
BT4
BT3
BT2
Part 2
![Page 21: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/21.jpg)
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
![Page 22: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/22.jpg)
Validation of Somatic Mutations in Genomic DNA
tumor
normal
P952R
ESYT1
tumor
normal
K217Q
RYBP
Part 2
![Page 23: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/23.jpg)
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
![Page 24: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/24.jpg)
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
![Page 25: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/25.jpg)
What Determine Allelic Variation in Gene Expression?
• Epigenetic mechanism
• Genetic mechanism
Part 3
![Page 26: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/26.jpg)
Allele-specific ChIP-on-chip Studies in Lymphoblastoid Cell Lines from CEPH Families
Part 3
![Page 27: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/27.jpg)
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
![Page 28: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/28.jpg)
RAS
A /(A+B)
Clustering of Samples Based on Family Using Allele-specific Chromatin Features
Part 3
![Page 29: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/29.jpg)
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
![Page 30: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/30.jpg)
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
![Page 31: Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ec65503460f94bd1d52/html5/thumbnails/31.jpg)
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