1. Investigations into Breast Cancer Etiology: Genomic/Epigenomic Analyses, Novel Oncogenes, and Allele-specific Studies Maxwell Lee National Cancer Institute Center for Cancer Research Laboratory of Population Genetics September 29 th, 2010

2. 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.

3. Our Approach to Understanding the Etiology of Breast Cancer breast tumors genomics cancer-related genes epigenomics Part 1 Mits Kadota

4. Clinical Information of Our Primary Breast Tumors (CHTN) A Numbers refer to tumor counts in each category Part 1

5. Primary Breast Tumors: DNA Copy Number Variation 161 tumors Part 1 chromosome 161 breast tumors putative novel oncogenes Affymetrix SNP5 array 1q 8q traditional approach our approach size of focal amplification multiple genes1 gene frequency of tumors with amplification common Don’t require common but require multiple occurrence

6. Focal Amplification of TBL1XR1 in Breast Tumors Part 1

7. Focal Amplification Detected in Primary Breast Tumors Part 1

8. Gene Amplification and Up-regulation at RNA and Protein Levels of TBL1XR1 in Breast Tumors focal amplification Hypothesis: additional mechanisms for up-regulation amplification or up-regulation DNA RNAprotein Part 1 genomic alteration RNA over-expression protein over-expression

9. Primary Breast Tumors: Frequent Over-expression of TBL1XR1 Well-diffpoorly-diff negative164 positive3515 negative 31% positive 69% N=84 odds ratio = 1.7 N=70 TBL1XR1 staining increases in poorly-differentiated tumors Part 1 In collaboration with Dr. Junya Fukuoka

10. 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 HRAS MCF10ATMCF10CA1h MCF10CA1a In collaboration with Dr. Lalage Wakefield

11. TBL1XR1-shRNA Knockdown Suppresses In Vivo Tumor Growth MCF10CA1hControl-shRNATBL1XR1-shRNA Tumor volume (mm 3 ) Day 39 N=10 N=14 N=5 N=7mice implants Part 1 Kadota et al. Cancer Research. 2009

12. Two Complementary Approaches Part 1 SNP array 10 kb resolution hundreds of tumors Functional studies Next-generation sequencing single nucleotide resolution a few samples Functional studies

13. 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.

14. Histopathological Data of Tumors sample tumor content ERPRHER2stagegradeinvasionnodetype BT1100--+T3N33yespositiveinvasive ductal carcinoma BT2100---T2N3c3yespositiveinvasive ductal carcinoma BT3100--+T33yesinvasive ductal carcinoma BT495---T2N03yesnegativeInvasive ductal carcinoma Part 2

15. Summary of Sequence Reads and BWA Mapping 76 base and 108 base pair-end sequences mapping pipeline Assemble bam files hg18 reference sequence mRNA refseq EST combination of any two exons Part 2 Million reads 20 40 60 140 120 100 80 splicing junction

16. The Number of Genes that Showed Differential Expression between Tumor and Normal Tissues tumornumber of genes that were down- regulated in tumor (FDR=0.1) number of genes that were up- regulated in tumor (FDR=0.1) BT1146023644 BT2138402905 BT3133277652 BT4125007584 Part 2

17. SOX10 Is Down-regulated in a Tumor chr22:36686044-36722706 BN1 BN4 BN3 BN2 BT1 BT4 BT3 BT2 normal tumor Part 2

18. MMP9 Is Up-regulated in Tumors chr20:44062819-44086029 triple negative BN1 BN4 BN3 BN2 BT1 BT4 BT3 BT2 triple negative Part 2

19. Comparison of Up-regulated Genes between Tumors BT1BT2BT3BT4 BT11005512 BT251001029 BT35101008 BT412298100 triple negative Part 2 2 triple negative tumors HER positive

20. An isoform of GNAS Is Down-regulated in Tumors mat pat bi-allelic BN1 BN4 BN3 BN2 BT1 BT4 BT3 BT2 Part 2

21. Somatic Mutation Summary BT1BT2 BT3BT4 tumor mutant reads > 10 normal mutant read = 0 tumor mutant fraction > 0.1 normal mutant fraction < 0.05 normal reference reads > 10 Part 2

22. Validation of Somatic Mutations in Genomic DNA tumor normal P952R ESYT1 tumor normal K217Q RYBP Part 2

23. Summary of Validation Experiments Part 2 genetumormutationmutant fractiondescription ESYT1BT4P952R1extended synaptotagmin-like protein 1 G3BP2BT2S48T0.2 GTPase activating protein (SH3 domain) binding protein 2; oncogene, sequesting TP53 GPRC5ABT2S59C0.4 G protein-coupled receptor, family C, group 5, member A; tumor suppressor in lung cancer INO80BBT1P306L0.2 INO80 complex subunit B; tumor suppressor in prostate cancer OSTF1BT3L20P0.2osteoclast stimulating factor 1 PIK3CABT3G1049R0.1phosphoinositide-3-kinase, catalytic, alpha polypeptide RAPH1BT2D1199N0.5 Ras association and pleckstrin homology domains 1 isoform 1; in JHU screen RYBPBT2K217Q0.5 RING1 and YY1 binding protein; tumor suppressor, stabilizing TP53 SHQ1BT4K193M0.5protein SHQ1 homolog USP6NLBT3V536I0.3USP6 N-terminal like VIMBT2E153A0.2vimentin Red highlights deleterious change by SIFT

24. 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 genes all or none parental origin Allelic gene expression 20%~50% genes quantitative difference sequence, cellular context, etc. > 2-fold difference 277 genes 326 genes Part 3

25. What Determine Allelic Variation in Gene Expression? Epigenetic mechanism Genetic mechanism Part 3

26. Allele-specific ChIP-on-chip Studies in Lymphoblastoid Cell Lines from CEPH Families Part 3

27. 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 active chromatin inactive chromatin Pat Mat A B DNA or LIT1, an imprinted gene Part 3

28. RAS A /(A+B) Clustering of Samples Based on Family Using Allele-specific Chromatin Features Part 3

29. Inheritance Analysis of Allelic Histone H3 Acetylation at the TMEM16D Locus Informative for SNP interrogated on SNP array (rs938335) RAS A/ (A + B) L: low H3Ac M: medium H3Ac H: high H3Ac L H HH MM Part 3 Kadota et al. PLoS Genet. 2007

30. Clustering of 42 Primary Breast Tumors by the Degree of Mono-allelic Methylation in Chromosome Arms A early stagelate stage triple neg. Part 3

31. Acknowledgments Mitsutaka Kadota Howard Yang Beverly Duncan Sheryl Gere Misako Sato Akira Ooshima Lalage Wakefield Robert Clifford Richard Finney Shuang Cai Chunhua Yan Michael Edmonson Daoud Meerzaman Ken Buetow Nan Hu Chaoyu Wang Hua Su Phil Taylor Shun-Ichiro KageyamaTakuya Nagata Junya FukuokaKazuhiro Tsukada NCI/CCR Kent Hunter Alisa Goldstein NCI/DCEG Barbara Dunn NCI/DCP Japan/ Toyama Univ. Jiuping Ji NCI/DCTD