Cancer regulators 2013


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Cancer regulators 2013

  1. 1. Identify the Key Players and Their Regulators in Major Cancer Types Jesse Liang, Ph.D. 1 Sample & Assay Technologies
  2. 2. 8 Hallmarks of Cancer . . . . . . . . . The 8 Hallmarks of Cancer (Cell, March 2011): 1. Sustaining proliferative signaling 2. Evading growth suppressors 3. Resisting cell death 4. Enabling replicative immortality 5. Inducing angiogenesis 6. Activating invasion and metastasis 7. Reprogramming energy metabolism 8. Escaping immune destruction -2- Sample & Assay Technologies
  3. 3. Corresponding PCR Arrays to Study These Hallmarks 1. Cell Cycle 2. Apoptosis 3. Cell Death Pathway Finder 4. Telomeres and Telomerases 5. Angiogenesis 6. Extracellular Matrix (ECM) and Adhesion Molecules 7. EMT (Epithelial-Mesenchymal Transition) 8. Tumor Metastasis 9. Cell Motility 10. Energy Metabolism 11. Innate and Adaptive Immune Response 12. Cancer Pathway Finder . . . . . . . . . . . . . -3- Sample & Assay Technologies
  4. 4. Profile Key Gene Expressions – Using RT2 Profiler PCR Array 84 Pathway-Specific Genes of Interest 5 Housekeeping Genes Genomic DNA Contamination Control Reverse Transcription Controls (RTC) Positive PCR Controls (PPC) -4- Sample & Assay Technologies
  5. 5. Simple Workflow of PCR Arrays cDNA Synthesis (kit) 45 minutes Load Plates (Use 8-Channel Pipettors) 2 minutes Run 40 cycle qPCR Program 2 to 2.5 hours compatible with all major real-time PCR instruments Upload and Analyze Data 15 minutes -5- Sample & Assay Technologies
  6. 6. Identify the Key Genetic Players in Breast Cancer . Breast Cancer PCR Array Breast cancer can be classified into normal breast-like, luminal, HER2-like, and basallike (also inaccurately called triple-negative) tumors. . Tumor Classification Markers: Luminal: ESR1 (ERa), FOXA1, GATA3, KRT8, KRT18, SLC39A6, TFF3, XBP1. HER2-like: ERBB2 (HER2), GRB7. Basal-like / Triple Negative: BIRC5, EGFR, KRT5, NOTCH1. Metastasis to Lung: ID1, MMP2 (Gelatinase A), PTGS2 (COX2). . Signal Transduction: Steroid Receptor-Mediated: AR, BRCA1, CCNE1, CTNNB1, ESR1 (ERa), ESR2 (ERß), IGF1, KRT19, PGR, RB1. Hedgehog: BCL2, CCND1, GLI1, SNAI2. Glucocorticoid: IGFBP3, NME1 (NM23A), NR3C1 (GRL). Classical WNT: APC, CCND1, CTNNB1, SFRP1. PI3K/AKT: AKT1, ERBB2 (HER2), IGF1, IGF1R, PTEN. NOTCH: BIRC5, NOTCH1. MAPK: MAPK1 (ERK2), MAPK3 (ERK1), MAPK8 (JNK1), TP73. . -6- Sample & Assay Technologies
  7. 7. Breast Cancer Subtypes Subtype These tumors tend to be Prevalence Luminal A ER+ and/or PR+, HER2-, low Ki67 42-59% Luminal B ER+ and/or PR+, HER2+ (or HER2with high Ki67) 6-19% Triple negative/basal-like ER-, PR-, HER2-, cytokeratin 5/6 + and/or HER1(EGFR)+ 14-20% HER2+ ER-, PR-, HER2+ 7-12% -7- Sample & Assay Technologies
  8. 8. Identify the Key Genetic Players in Lung Cancer . Lung Cancer PCR Array non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC) . Differentially Methylated Promoters: APBA1, APC, CADM1, CDH1, CDH13, CDKN1C, CDKN2A, CDKN2B, CXCL12, CYP1B1, DLC1, FHIT, MGMT, MLH1, MTHFR, OPCML, PRDM2, RASSF1, RASSF2, SFRP1, TCF21 Metastatic Potential: ANXA5, DUSP6, ERBB3, HGF, HMMR, IRF4, LCK, MET, MMD, NF1, STAT1, STAT2 . . Differentially Expressed Genes in Adenocarcinoma (AC) Versus Squamous Cell Carcinoma (SCC): Higher in AC than in SCC: CLCA2, DSG3, KRT5, KRT14, SPRR1A Higher in SCC than in AC: AGR2, LGSN, NKX2-1, SFTA3, SPINK1 . . Up-Regulated in Lung Cancer: CEACAM5, COL11A1, CP, CXCL13, GREM1, MKI67, MMP1, MMP12, SPINK1, SPP1, TOP2A, TOX3 Down-Regulated in Lung Cancer: AGER, CA4, CLDN18, CLIC5, FABP4, GPM6A, SCGB1A1, SFTPC, SOSTDC1, WIF1 . . -8- Sample & Assay Technologies
  9. 9. Identify the Key Genetic Players in Prostate Cancer . Prostate Cancer PCR Array . Differentially Methylated Promoters: APC, AR, CAV1, CCNA1, CDH1, CDKN2A, DKK3, DLC1, EDNRB, GPX3, GSTP1, MGMT, MSX1, PDLIM4, PTGS2, RARB, RASSF1, SFRP1, SLC5A8, TIMP2, TNFRSF10D, ZNF185 . Up-Regulated in Prostate Cancer: ARNTL, CAMSAP1, DDX11, ECT2, ETV1, HAL, IGFBP5, KLK3, MTO1, PDPK1, RBM39, SOCS3, SOX4, SUPT7L . Down-Regulated in Prostate Cancer: CCND2, CLN3, GCA, IGF1, LGALS4, LOXL1, PPP2R1B, SFRP1, SLC5A8, TFPI2, USP5 . Metastatic Potential: CREB1, INTS6, KLHL13, MAX, NDRG3, PES1, SEPT7, SCAF11 . Androgen Signaling: AR, CAV1, CCND1, DAXX, EGFR, FOXO1, GNRH1, IGF1, IL6, NFKB1, NRIP1, PTEN, SHBG, TGFB1I1, TIMP2, TIMP3, VEGFA . -9- Sample & Assay Technologies
  10. 10. Identify the Key Genetic Players in Liver Cancer . Liver Cancer (HCC) PCR Array . Genes Up-Regulated During HCC: BCL2, BIRC5, CCND1, CFLAR (CASPER), FZD7, IGF2, IRS1, LEF1, MCL1, MET, NFKB1, PIN1, PTK2, RAC1, RHOA, SMAD7, STAT3, TCF4, TGFA, TGFB1, TNFSF10 (TRAIL), XIAP . Genes Down-Regulated During HCC: BAX, BID, CASP8 (FLICE), CDKN1A (p21CIP1/WAF1), CDKN2A (p16INK4), DLC1, FADD, FAS (TNFRSF6), HHIP, IGFBP1, IGFBP3, ITGB1, PTEN, RB1, SMAD4, SOCS1, SOCS3,TGFBR2, TNFRSF10B (DR5) . Signal Transduction: Classical WNT: CCND1, CDKN2A (p16INK4), CTNNB1, FZD7, LEF1, MTDH, PIN1, RB1, SFRP2, TCF4. TGFß: SMAD4, SMAD7, TGFB1, TGFBR2 PI3K/AKT: AKT1, IRS1, PTEN. IGF/IGFR: IGF2, IGFBP1, IGFBP3, IRS1 Hippo: YAP1 (Yes-Associated Protein (YAP) is a potent oncogenic driver and an . independent prognostic risk factor of HCC) Ras/Raf/MEK/ERK: CDKN1A (p21CIP1/WAF1), CDKN2A (p16INK4), DLC1, HRAS, NRAS, RAC1, RASSF1, RB1, RHOA, TP53 EGFR: ADAM17 (CD156B), CDH13, EGF, EGFR, TGFA, TGFB1 MET/HGF: MET, HGF Small GTPase-Mediated: CDH13, CDKN1A (p21CIP1/WAF1), CDKN2A (p16INK4), DAB2IP, DLC1, HRAS, NRAS, RAC1, RASSF1, RB1, RELN, RHOA, TP53 - 10 - Sample & Assay Technologies
  11. 11. Identify the Key Genetic Players in Leukemia . Leukemia PCR Array Clinical and pathological subtypes of leukemia define both its onset (acute or chronic) and the affected blood cell type (lymphoblastic/lymphocytic or myeloid/myelogenous). . . Differentially Methylated Promoters: APAF1, CADM1, CDH1, CDH13, CDKN1A, CDKN1B, CDKN1C, CDKN2B, DAPK1, DKK3, DLC1, EGR3, HIC1, JUNB, MEIS1, MLH1, NPM1, PTEN, SFRP2, SFRP4, SFRP5, SOCS1, TP53, WIF1 BCR-ABL Pathway: ABL1, BCR, GRB2, JAK2, SHC1, STAT3, STAT5A, STAT5B JAK-STAT Signaling: AKT1, CSF3, IFNA1, IL4, IL6, JAK2, STAT1, STAT3, STAT5A, STAT5B, SOCS1 Hemopoiesis: BCL2, CSF3, CTNNB1, IL4, JAK2, LMO2, PML, RUNX1, STAT5A, STAT5B, TAL1, TP53 Myeloid Cell Differentiation: CTNNB1, FOXO3, IL4, JUN, LMO2, RUNX1, SPI1, STAT5A, STAT5B, TAL1 Immune System Development: BCL2, CSF3, CTNNB1, IL4, JAK2, LMO2, MLH1, PML, RUNX1, STAT5A, STAT5B, TAL1, TLX1, TP53 Lymphocyte Proliferation/Differentiation: BCL2, CSF3, CTNNB1, IL4, IL6, STAT5A, STAT5B Cytokines & Chemokines: CCL3, CSF3, CXCL10, IFNA1, IL1R1, IL4, IL6, IL8, IL10, IL12A Malignancy: BAALC, BMPR1B, BTG3, CD34, CDC42EP3, CTGF, GAS2L3, MN1, NR4A3, RGS12 Leukemia Therapeutic Targets: AKT1, ALOX5, BCL2, CTNNB1, FGR, FOXO3, HCK, HDAC1, HSP90AA1, IFNA1, JAK2, MCL1, MERTK, MTOR, PML, PRKCB, RAC2, SMO Leukemia Stem Cell Transcriptional Regulators: CEBPB, GATA1, IKZF3, JUN, LMO1, LMO2, LYL1, MEIS1, NFKB1, RUNX1, RUNX2, RUNX3, SPI1, STAT1, STAT3, STAT5A, TAL1, TLX1, TLX3 . . . . . . . . . . . - 11 - Sample & Assay Technologies
  12. 12. Identify the Key Genetic Players in Lymphoma Lymphoma PCR Array . . . . . . . . . . . . . Differentially Methylated Promoters: AFF1, ATM, CADM1, CCND1, CDH1, CDH13, CDKN1C, CDKN2B, CDKN2C, DAPK1, DLC1, FHIT, GSTP1, HIC1, HRK, ID4, LRP1B, MLH1, MYOD1, RASSF1, RBP1, SLC19A1, TIMP2, TIMP3 DLBCL (Diffuse Large B-cell Lymphoma) Survival: ASB13, BCL6, ITPKB, LMO2, LRMP, MYBL1, PAG1, PTK2, TTC9, VGLL4 Follicular Lymphoma Survival: C1QA, C4A, CCR1, F8, FCGR1A, FLT3LG, STAT4, TLR5, TNFRSF1B, TNFSF13B Lymphoma Therapeutic Targets: BCL6, BIRC5, BTK, CD22, CD40, CD80, CXCR4, FCGR1A, HSP90AA1, MCL1, MS4A1, MTOR, NOTCH1, PARP1, PTK2, SRC, SYK, TGFB1, TNFSF13B Inflammatory Response: C1QA, C4A, CCR1, CD40, CD40LG, CSF3, CXCR4, F8, IL6, NFKB1, TGFB1, TLR5, TNF, TNFRSF1B Immune System Process: BCL6, C1QA, C4A, CADM1, CD2, CD4, CD40, CD5, CD79A, CD80, FCER2, IFNG, IL2, IL6, ITPKB, PDLIM7, PTPRC, SYK, TGFB1, TNFSF13B T Cell Differentiation: BCL2, CD3D, CD4, CD8A, ITPKB, IL2, PTPRC, SYK, TGFB1, TP53 Cell Adhesion: BCL2, CADM1, CCR1, CD2, CD22, CD34, CD4, CD40LG, CD8A, CDH1, CDH13, DLC1, F8, IL2, NCAM1, PTPRC, SRC, SYK, TNF Cell Cycle: ATM, BCL2, BIRC5, CCND1, CDKN1C, CDKN2B, CDKN2C, ID4, IFNG, MKI67, MLH1, MYC, RASSF1, TGFB1, TP53 PI3K/AKT Signaling: ALK, BCL2, BTK, CCND1, CCR1, CD2, CD4, CD40, CD40LG, CSF3, CXCR4, HSP90AA1, IL2, IL6, MTOR, PTK2, SYK, TGFB1, TIMP2, TIMP3, TNFRSF1B, TNFSF13B, TP53 Gene Fusion: ALK, MME DNA Repair: DNTT - 12 - Sample & Assay Technologies
  13. 13. Methylation PCR Arrays . . 1. DNA Digestion Genomic DNA sample is aliquoted into four equal portions: a. Mock Digest (Mo) No enzymes are added in this reaction. The product of the mock digestion represents the total amount of input DNA for real-time PCR detection. b. Methylation Sensitive Digest (Ms) Cleavage with a methylation-sensitive enzyme will digest unmethylated and partially methylated DNA. The remaining (hyper)-methylated DNA will be detected by real-time PCR. c. Methylation Dependent Digest (Md) Cleavage with a methylation-dependent enzyme will preferentially digest methylated DNA. The remaining unmethylated DNA will be detected by realtime PCR. d. Double Digest (Msd) Both enzymes are added in the double digest, and all DNA molecules (both methylated and unmethylated) will be digested. This reaction measures the background and the fraction of input DNA refractory to enzyme digestion. . . . . . . . . . . . . 2. Real-Time PCR 3. Data Analysis We provide user-friendly free online data analysis tool. - 13 - Sample & Assay Technologies
  14. 14. What Methylation PCR Array Offers – a Quick Screening Tool . What Does Methylation PCR Array Offer? . . Disease or Pathway Focused: Simultaneously detect DNA methylation of 24-96 genes on a 96-well or 384-well plate. . Genome-Wide Coverage: DNA methylation analysis of any human, mouse or rat genes. You can also order individual primers for an individual gene. Ordering custom array is also welcome. . Easy, Fast & Reliable: Simple restriction enzyme digestion followed by optimized qPCR. No bisulfite conversion, cloning or sequencing. - 14 - Sample & Assay Technologies
  15. 15. Identify the Key Epigenetic Players in Breast Cancer Breast Cancer Methylation PCR Array: Cell Cycle, Growth, Differentiation & Development: BRCA1, CCNA1, CCND2, CDKN1C, CDKN2A (p14ARF), CDKN2A (p16), SFN (14-3-3 sigma), TP73 Cell Adhesion: CDH1, CDH13 Transcription Factors: CDKN2A (p14ARF), ESR1, HIC1, PRDM2, RASSF1, TP73 Hormone Receptors: ESR1 Drug Metabolism: GSTP1 Apoptosis & Anti-Apoptosis: CDKN2A (p14ARF), CDKN2A (p16), PYCARD, TNFRSF10C, TNFRSF10D, TP73 DNA Methylation: MGMT, PRDM2 Phosphatases: PTEN Prostaglandin-Endoperoxide Synthase: PTGS2 Extracellular Matrix Molecules: ADAM23, SLIT2, THBS1 Proteases & Protease Inhibitors: DAM23, THBS1 Ras Associated Protein Superfamily: RASSF1 - 15 - Sample & Assay Technologies
  16. 16. Identify the Key Epigenetic Players in Lung Cancer Lung Cancer Methylation PCR Array: . Apoptosis & Anti-Apoptosis: APC, CADM1 (TSLC1), CDKN1C (p57Kip2), CDKN2A (p14ARF), CDKN2B (p15INK4b), FHIT Cell Adhesion & Migration: APC, CADM1 (TSLC1), CDH1, CDH13, DLC1, OPCML (aOBCAM) Cell Cycle, Growth, Differentiation & Development: APC, CADM1 (TSLC1), CDKN1C (p57Kip2), CDKN2A (p14ARF), CDKN2B (p15INK4b), DLC1, FHIT, MLH1, PRDM2 (RIZ1), RASSF1, RASSF2, TCF21 Chemokine: CXCL12 Neuronal Adaptor Protein: APBA1 (MINT1) DNA Damage Repair: APC, MLH1, RASSF1 DNA Methyl Transferase: MGMT (AGT), PRDM2 Drug Metabolism: CYP1B1 Oncogene: PAX5 Histidine Triad Gene Family: FHIT miRNA Genes: has-let-7a, hsa-miR-124 One Carbon Unit Metabolism: MTHFR Transcription Factors: PAX5, PRDM2 (RIZ1), TCF21 Tumor Suppressor Gene Candidates: APC, CDKN1C (p57Kip2), CDKN2A (p14ARF), CDKN2B (p15INK4b), MLH1, RASSF1, RASSF2 WNT Signaling: APC, SFRP1 . . - 16 - Sample & Assay Technologies
  17. 17. Identify the Key Epigenetic Players in Prostate Cancer Prostate Cancer Methylation PCR Array: . Apoptosis & Anti-Apoptosis: APC, CDKN2A (P16), TNFRSF10D Calcium Channel Signaling: EDNRB Caveolae Protein: CAV1 Cell Cycle, Growth, Differentiation & Development: APC, CCNA1, CDKN2A (P16), DLC1, RARB, RASSF1 Cell Adhesion & Migration: APC, CDH1, DLC1, OPCML (aOBCAM), TIMP2 Cytokine: TNFRSF10D Drug Metabolism: GPX3, GSTP1, SLC5A8 DNA Methyltransferase: MGMT (AGT) Extracellular Matrix Remodeling: TIMP2 Inflammation: PTGS2 (COX2) miRNA Genes: hsa-miR-34a Mitogenesis: PTGS2 (COX2) Nuclear Receptors: AR, RARB Oncogene: CAV1 Protease Inhibitor: TIMP2 Retinoid Acid Signaling: RARB Solute Carrier Family: SLC5A8 Transcription Factors and Cofactors: MSX1, PDLIM4 (RIL) Tumor Suppressor Gene Candidates: APC, CAV1, CDKN2A (P16), RASSF1, SLC5A8, ZNF185 Wnt Signaling Pathway: APC, DKK3, SFRP1 . - 17 - Sample & Assay Technologies
  18. 18. Identify the Key Epigenetic Players in Liver Cancer Liver Cancer Methylation PCR Array: . . Angiogenesis: EP300 Apoptosis & Anti-Apoptosis: CDKN1B, CDKN2A (P16,), FHIT, PYCARD (TMS1/ASC), TNFRSF10D Cell Adhesion & Migration: CDH1, DLC1, OPCML (aOBCAM), RELN Cell Cycle, Growth, Differentiation & Development: CCND2 (Cyclin D2), CDKN1A, CDKN1B (p27KIP1), CDKN2A (P16), DLC1, EP300, FHIT, RASSF1, WT1 Cytokine Receptor: TNFRSF10D Cytokine Signaling Inhibitor: SOCS1 DNA Damage Repair: CDKN1A, MSH2, MSH3, RASSF1 Drug Metabolism: GSTP1 Extracellular Matrix Remodeling: RELN GTPase-Activating Protein: DAB2IP Histidine Triad Gene Family: FHIT Inflammation: PYCARD (TMS1/ASC) miRNA Gene: hsa-miR-1 Oncogene: E2F1 Transcription Factors: E2F1, EP300, RUNX3, WT1, EP300 Tumor Suppressor Gene Candidates: DLEC1, CDKN1A, CDKN2A (P16), E2F1, MSH2, MSH3, RASSF1, RUNX3 Wnt Signaling Pathway: SFRP2 . - 18 - Sample & Assay Technologies
  19. 19. Identify the Key Epigenetic Players in Leukemia & Lymphoma Leukemia & Lymphoma Methylation PCR Array: . . Development & Differentiation: HCK, MAFB, MEN1, TP53 Cell Cycle: JUNB, MEN1, NPM1, TP53 DNA Damage & Repair: FANCC, FANCL, NPM1, PMS1, TP53 Extracellular Matrix and Adhesion: CD9, CTNNA1, SPOCK2 Growth & Proliferation: DRD2, MEN1, NPM1, SLC5A8, TP53 Signal Transduction: DRD2, EXT1, MEN1, NFATC1, NPM1, PARK2, PER2, TLE1, TP53 Transcription Factors & Co-Factors: AFF1, CEBPD, HOXA7, HOXB5, JUNB, MAFB, NFATC1, NPM1, TLE1, TP53 Other: LMNA . . . . . . . . - 19 - Sample & Assay Technologies
  20. 20. Identify the Key Epigenetic Players in Other Cancers Colon Cancer: . . Gastric Cancer: . . Melanoma: . -------------------------------------------------------------------------------------------------------------- . . EMT (Epithelial-Mesenchymal Transition): . . Tumor Suppressor Genes: . . Apoptosis: . . Cell Cycle: . . DNA Repair: . . Cancer miRNA: . . - 20 - Sample & Assay Technologies
  21. 21. Important Note . Please Note: If you study lung cancer, for example, I greatly recommend that you use regular Lung Cancer PCR Array and Methylation Lung Cancer PCR Array to get corresponding results. This applies to other cancer types, also to apoptosis, cell cycle, DNA damage, EMT, tumor suppressors, and cancer-related miRNAs researchers. . - 21 - Sample & Assay Technologies
  22. 22. Cancer Mutation PCR Arrays and Assays Custom available! We also provide over 900 individual gene assays: - 22 - Sample & Assay Technologies
  23. 23. Overview 45-Signaling Pathway Activity in a Cell Assay Transcription factor activity can be used as a readout for the activation status of signal transduction pathways. Therefore, the development of reliable cell-based assays to measure transcription factor activity is a crucial technology for fast and efficient global functional genomics and chemical genetics studies. . . Cignal 45-Pathway Reporter Array is a novel cell-based assay for monitoring 45 cell signaling pathways in cultured cells in a single experiment. You can observe phenotypes and quantitate as a dual-luciferase assay. For individual assay: - 23 - Sample & Assay Technologies
  24. 24. We Provide Service – Send Samples to Us & Receive Results! Whole Genome Illumina Gene Expression Profiling Illumina Genotyping . Pathway / Focused Panel Mutation Profiling Methylation PCR Array miRNA PCR Array . . . . . Individual Gene / Locus Mutation Detection Methylation qPCR . . . . Sample Preparation – DNA, RNA Extraction and Purification Cells, Tissue or Biofluids Fixed Tissue Small Sample . . . . . - 24 - Sample & Assay Technologies
  25. 25. Contact Information . . . . . Jesse Liang Email: Technical Support: 1-888-503-3187 Email: Check Webinar Calendar: . - 25 - Sample & Assay Technologies