Cancer Genomics (Current technologies & clinical implication)

Transcription

1 Cancer Genomics (Current technologies & clinical implication) Jeonghee Cho, Ph.D. Samsung Medical Center Samsung Cancer Research Institute

2 Robert Weinberg (M.I.T) Douglas Hanahan (ISREC)

3 The Hallmarks of Cancer

4 1) Inducing and sustaining proliferative signaling - The most fundamental trait of cancer cells (lack of homeostasis) - Deregulation of growth factors mediated signalings : Production of growth factor ligands autocrine proliferative stimulation : Elevating the levels of receptor proteins at the cancer cell surface : Receptor structural change constitute activation of the receptor : Constitute activation of components of signaling pathways downstream - Somatic mutation activate additional downstream pathways : ~40% of human melanoma B-Raf mutation : ~30% NSCLC EGFR mutation - Disruptions of Negative-Feedback Mechanisms : Ras mutation, PTEN loss - Excessive proliferative signaling can trigger cell senescence : Intrinsic cellular defense mechanism

5 2) Evading Growth Suppressors - Cancer cells must circumvent powerful programs that negatively regulate cell proliferation; actions of tumor suppressor genes - RB (retinoblastoma-associated) : A critical gatekeeper of cell-cycle progression : Lack of RB function induces persistent cell proliferation - TP53 : abnormality sensors within the cell s intracellular operating system (levels of nucleotide, glucose, DNA damage) - Evasion of contact inhibition, - Corruption of the TGF-beta signaling pathway

6 3) Resisting Cell Death (apoptosis) - The concept that programmed cell death by apoptosis serves as a natural barrier to cancer development. - Loss of tumor suppressor genes (ex)rb, TP53) - Overexpression of antiapoptotic or Downregulation of proapoptotic proteins - Necrosis has proinflammatory and tumor-promoting potential : necrotic cell death releases proinflammatory signals into the surrounding tissue microenvironment. - Autophagy mediates both tumor cell survival and death : Induction of autophagy can serve as a barrier to tumorigenesis (early stage)

7 4) Enabling Replicative Immortality - Senescence: a typically irreversible entrance into a nonproliferative but viable state - Crisis: cell death - Telomerase: the specialized DNA polymerase that add telomere repeat segments to the ends of telomeric DNA - The eventual immortalization of rare variant cells that proceed to form tumors has been attributed to their ability to senescence or apoptosis, achieved most commonly by upregulating expression of telomerase or, less frequently, via an alternative recombination-based telomere maintenance mechanism.

8 5) Activating Invasion and Metastasis - Downregulation of cell-to-cell adhesion molecules (E-cadherin) potentiate invasion and metastasis - Epithelial-mesenchymal transition (EMT) is a mean by which transformed epithelial cells can acquire the abilities to invade, to resist apoptosis, and to disseminate.

9 Emerging Hallmarks and Enabling Characteristics

10 Intracellular Signaling Networks Regulate the Operations of Cancer Cell

11 Signaling interaction in the Tumor Microenvironment during Malignant Progression

12 Therapeutic target of the Hallmarks of Cancer

13

14 Cancer is a Genetic Disease Current model of cancer development Somatic Alterations of the Genome Altered Function or Expression of Oncogenes/ Tumor Suppressor Genes Cancer Cell Phenotypes Point Mutations Copy Number Alterations Chromosomal Rearrangements Epigenetic Modifications Infections Uncontrolled proliferation Prolonged survival Immortalization Angiogenesis Invasion/Metastasis Hanahan and Weinberg, Cell (2000)

15 Why Study Cancer Genomes? 1. Discover novel genes and pathways important for cancer development 2. Develop a natural classification scheme for human cancers 3. Identify targets for therapeutic intervention Example: Activating point mutations in EGFR kinase domain in a subset of NSCLC patients predicts sensitivity to EGFR kinase inhibition Paez et al, Science (2004); Lynch et al, NEJM (2004) Mok et al,nejm,(2009)

16 Structural Alterations

17 SNP genotypes in the presence of CNVs. LaFramboise T Nucl. Acids Res. 2009;37: ( Author(s 2009 The

18 Types of structural variant Copy-number variant (CNV). A segment of DNA that is 1 kb or larger and is present at a variable copy number in comparison with a reference genome. Classes of CNVs include insertions, deletions and duplications. This definition also includes large-scale copy-number variants, which are variants that involve segments of DNA 50 kb, allowing them to be detected by clone-based array comparative genome hybridization (array-cgh). Segmental duplication or low-copy repeat. A segment of DNA >1 kb in size that occurs in two or more copies per haploid genome, with the different copies sharing >90% sequence identity. They are often variable in copy number and can therefore also be CNVs. Inversion. A segment of DNA that is reversed in orientation with respect to the rest of the chromosome. Pericentric inversions include the centromere, whereas paracentric inversions do not. Translocation. A change in position of a chromosomal segment within a genome that involves no change to the total DNA content. Translocations can be intra- or inter-chromosomal.

19 Cytogenetic detection and conformation of structural variants Feuk et al. Nature Reviews Genetics 7, (February 2006) doi: /nrg1767

20 Methods for detecting structural variants in the human genome Feuk et al. Nature Reviews Genetics 7, (February 2006) doi: /nrg1767

21 Array-based, genome-wide methods for the identification of copy-number variants Feuk et al. Nature Reviews Genetics 7, (February 2006) doi: /nrg1767

22 Multiplex PCR-based methods for the identification of copy number variants Feuk et al. Nature Reviews Genetics 7, (February 2006) doi: /nrg1767

23 Influence of structural variants on phenotype Feuk et al. Nature Reviews Genetics 7, (February 2006) doi: /nrg1767

24 The dataset: 250K SNP arrays across 26 major types # of samples 2528 tissue samples 603 cell lines 3131 Total Top 25 subtypes Beroukhim, Mermel et al, Nature (2010)

25 Study of focal SCNAs afford greater opportunity to identify targeted genes Total 82 regions Total 76 regions Median 7 genes/ 95% confidence region 70 regions without known target Median 6.5 genes/95% confidence region 52 regions without known target Most of the top 20 significantly amplified and deleted regions across cancer are already known However, the 9 th most significant focal amplification is a region on 1q containing 9 genes including MCL1 Beroukhim, Mermel et al, Nature (2010)

26 Characterizing the cancer genome in lung adenocarcinoma. Weir, B et al. Nature 2007

27 SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas Bass, A et al. Nature Genetics 2009

28 Expression Profiles

29 Expression profiling using cdna microarrays

30 RGB overlay of Cy3 and Cy5 images

31

32 Gene Expression profile identify four gene expression subtypes Survival by treatment and tumor subtype

33 ncounter Instrumentation

34 NanoString ncounter gene expression system Nature Biotechnology 26, (2008) 이시스템에최대장점은 mrna 를 cdna 로만들지않고적응 RNA(100ng-200ng) 을이용하여 expression 확인이가능 하다는점입니다. PCR 과정에서과도하게증폭되어증폭된유전자들때문에적은양을가지는유전자들이마스킹되 는경우가많이발생하나이시스템은 PCR 과정이없기때문에더정확한유전자의 expression 이확인가능합니다. 이시스템은 capture probe 와 reporter probe 두가지가목적유전자를유전자서열특이적으로결합하게되는원리 이고 Reporter probe 에는 labeled rna segments 들이존재하여그유전자가무엇이지알수있는바코드역할을합니 다.

35 1. Target(mRNA) 를 capture probe 와 reporter probe 가유전자서열특이적으로결합. 이때 reporter probe 에는이유전자가무엇인지알수있는 barcod(6 개의동그라미 ) 가붙어있다. 2, mrna 에붙지못한 capture probe 와 reporter probe 는제거된후 cartridge 에전기적반응으로심어진다. 3. Cartridge를스캐너를이용하여 color를읽는다. 옆에보시면는 3개가있고이유전자는 XLSA 이다. 출처 : Nanostring 홈페이지

36 NanoString ncounter mirna expression system - 이전의 gene epxression 과동일한원리이며, 단 mature mirna 와 mirtag sequence 가 Bridge 를이용한 ligation 과정을통해결합된다. 그후 bridge 는제거되고결합된 mature mirna 와 mirtag sequence 를 probe 가인식하여진행된다. 출처 : Nanostring 홈페이지

37 Mutation Analysis

38 EGFR mutations in Lung Cancer Exon 21 41% Exon 18 5% Exon 19 48% Somatic EGFR mutations found in ~30% of East Asian lung adenocarcinomas, ~7-10% of U.S. and European patients EGFR kinase domain mutations Exon 20 6% Frequency of mutation by exon Clustered in four areas o Nucleotide binding (P loop), G719 o Exon 19 deletions o Exon 20 insertions (C-terminal to alpha helix) o Activation loop mutations (L858) Mutation status predictive of clinical response to gefitinib, erlotinib o Ex19 deletions o L858R Paze et al Science

39 Ilumina sequencing animation HcxC4

40 The mutational landscape of head and neck squamous cell carcinoma. Stransky, N et al Science

41 Graphical representation of seven prostate cancer genomes M F. Berger et al. Nature 470, (2011) doi: /nature09744

42 Complex structural rearrangements in prostate cancer M F. Berger et al. Nature 470, (2011) doi: /nature09744

43 Disruption of CADM2 and the PTEN pathway by rearrangements M F. Berger et al. Nature 470, (2011) doi: /nature09744

44 - Targeted sequences : 441 tumors (1,507 coding genes) : 2,576 somatic mutations found -Whole-exome sequencing -Gene copy number analysis : 32 primary tumors : 911 somatic mutations (725 genes) -Whole-exome sequencing : 74 tumor-normal match : 321 somatic mutations (coding)

45 Functional Genomics

46 Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling Wagle N et al. JCO 2011;29: Kinase oncogene dependence and principles of drug resistance

47 Drug target discovery Cancer Genomic characterization -Copy number analysis -Expression analysis -Whole-genome, whole-exome sequencing Candidate target discovery (too many!!) High-Throughput Functional target validation -Gene knockdown screening (shrna) -Gene overexpression screening (ORFs) -Driver & Passenger -Drug resistant modulator -Combinatorial therapy Drug Development & Clinical application

48 RNAi 기법을이용한유전자기능연구 RNAi 는 dsrna 에의해서열특이적으로 mrna 가분해되며, 그결과유전자발현이억제되는현상임 RNAi 는유전자기능및형질변화연구에절대적으로필요함 (10,349 papers in Medline) 여러 RNAi 방법중 shrna lentiviral vector 가가장효율적인것으로알려져있음 (663 papers in Medline) RNAi 기법을통한체계적유전자발현저하를이용, 각유전자의기능연구 필요 : RNAi 를통한특정유전자발현저하 세포의형질변화? 각각의유전자타겟에필요한 RNAi reagents 유전자기능연구를위한세포및동물모델시스템 세포의형질변화에대한정확한판독

49 High-throughput shrna screening 방법 Arrayed Screens Hypothesis driven gene sets Robust interrogation of each gene High Content Screening Pooled Screens Genome-scale screening Many conditions: drug doses, time points, cell types etc. Long time course assays - proliferation and survival Lower resolution readout is differential representation of hairpin measured by microarray or sequencing

50 Pooled RNAi screening strategy and performance using pools of 45,000 shrna-expressing viruses. Luo B et al. PNAS 2008;105:

51 shrna-mediated drug target discovery PAX8 is essential for ovarian cancer cell proliferation and survival. Cheung H W et al. PNAS 2011;108:

52 Open Reading Frames (ORFs) expression 16,000여개의 coding ORFs와중요한 non-coding ORFs를이용한특정단백질의 over-expression 기법은특정유전자의발현을저해하는 RNAi 기법과상보적으로유전자의세포내기능을연구하는데주요하게사용될것임 ORF donor library L1 L2 Viral-delivery ORFs library Promoter element Structural studies N-terminal tag C-terminal tag Biochemical characterization/purification ORF Expression clones Protein localization Protein-protein interactions

53 An ORF-based functional screen identifies COT and C-RAF kinases as drivers of resistance to B-RAF inhibition. CM Johannessen et al. Nature 000, 1-5 (2010) doi: /nature09627

54 COT expression predicts resistance to B-RAF inhibition in cancer cell lines. CM Johannessen et al. Nature 000, 1-5 (2010) doi: /nature09627

55 COT-expressing B-RAF(V600E) cell lines exhibit resistance to allosteric MEK inhibitors. CM Johannessen et al. Nature 000, 1-5 (2010) doi: /nature09627