Metabolism and cancer 2013

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Metabolism and cancer 2013

  1. 1. Sample & Assay Technologies Metabolism and cancer
  2. 2. Sample & Assay Technologies Biology-focused solutions for pathway analysis • SABiosciences is now a Company Metabolism and cancer, Frederick, MD, USA, 11/14/12 2
  3. 3. Sample & Assay Technologies Metabolism & cancer: A brief history 1923: Otto Warburg proposed that cancer cells metabolize glucose differently from normal cells Aerobic glycolysis Pyruvate converted to lactate instead of entering the TCA cycle Common mechanism for cancer cells This key observation was ignored by the majority of scientists for decades Undergoing a “renaissance” in the past 10 years Warburg effect Oncogenes and tumor suppressor genes can alter cellular metabolism Metabolism and cancer, Frederick, MD, USA, 11/14/12 3
  4. 4. Sample & Assay Technologies Metabolic pathway overview Warburg effect Levine, A.J. (2010) Science 330, 1340. Metabolism and cancer, Frederick, MD, USA, 11/14/12 4
  5. 5. Sample & Assay Technologies Metabolic regulators overview Many oncogenes and tumor suppressor genes regulate metabolism Munoz-Pinedo, C. (2012) Cell Death & Dis. 3, e248. Metabolism and cancer, Frederick, MD, USA, 11/14/12 5
  6. 6. Sample & Assay Technologies Metabolism & cancer experimental design QIAGEN offers a variety of products for metabolism & cancer Mutational analysis Somatic mutation Copy number variation (CNV) Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Metabolism and cancer, Frederick, MD, USA, 11/14/12 6
  7. 7. Sample & Assay Technologies Somatic mutation analysis: Overview Hydrolysis probe + ARMS (Amplification Refractory Mutation System) Common Region F F Metabolism and cancer, Frederick, MD, USA, 11/14/12 Q Q 7
  8. 8. Sample & Assay Technologies Key metabolic mutations in cancer Oncogenes TS Genes Epigenetics IDH 2-HG Modified from: Deberardinis, R.J. (2008) Genet. Med. 10, 767. Metabolism and cancer, Frederick, MD, USA, 11/14/12 8
  9. 9. Sample & Assay Technologies Somatic mutation: TCA cycle IDH1 and IDH2 are currently hot topics Metabolism and cancer, Frederick, MD, USA, 11/14/12 9
  10. 10. Sample & Assay Technologies Somatic mutation: TCA cycle IDH1 R132H shows altered metabolic activity. Reitman, Z.J. (2010) JNCI. 102, 932. Metabolism and cancer, Frederick, MD, USA, 11/14/12 10
  11. 11. Sample & Assay Technologies Copy Number Variation (CNA/CNV) overview Discovery • • • Validation Array CGH SNP Chips NGS • • Diagnostic test FISH qPCR Metabolism and cancer, Frederick, MD, USA, 11/14/12 11
  12. 12. Sample & Assay Technologies CNV: Cancer & metabolism Methods: 42 serous ovarian cancer samples + 379 cancer samples from the Cancer Genome Atlas (http://tcga.cancer.gov/). CNV detection via representation oligonucleotide microarray analysis (ROMA). Correlated with gene expression via microarray. Results: Identified 346 genes with significant amplifications or deletions; 156 correlated with gene expression alterations. Metabolism and cancer, Frederick, MD, USA, 11/14/12 12
  13. 13. Sample & Assay Technologies CNV: Cancer & metabolism Of these 156 genes, 16 are relevant for metabolism: Carbohydrate metabolism AKR1A1 (Oxidoreductase) GPI (Isomerase) PRKACA (Kinase) Lipid metabolism CEBPA (Transcription factor) CPNE3 (Transporter) ECH1, EBP (Isomerases) Oxidative phosphorylation COX6B1, NDUFC2, NDUFS5, UQCRB, UQCRFS1 Amino acid metabolism CTPS (Synthase) PEPD (Peptidase) Oncogenes/Tumor suppressor genes KRAS MYC Metabolism and cancer, Frederick, MD, USA, 11/14/12 13
  14. 14. Sample & Assay Technologies Metabolism & cancer experimental design QIAGEN offers a variety of products for metabolism & cancer Mutational analysis qPCR detection Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Metabolism and cancer, Frederick, MD, USA, 11/14/12 14
  15. 15. Sample & Assay Technologies Principles of qRT-PCR: Overview Real-time PCR Amplify and simultaneously quantify target DNA Reverse transcription real-time PCR Amplify and simultaneously quantify mRNA Ct values: Threshold cycle Metabolism and cancer, Frederick, MD, USA, 11/14/12 15
  16. 16. Sample & Assay Technologies Gene expression: Serotonin metabolism Is serotonin metabolism central to cholangiosarcoma growth? Background: Cholangiosarcoma is a hepatic cancer with few treatment options. Serotonin is normally secreted from cholangiocytes. Opposing effects of serotonin with various cancers have been reported Methods: Grow multiple cholangiosarcoma cell lines Examine serotonin metabolism via direct measurement and gene expression of metabolic pathways Confirm hypothesis with nude mice Metabolism and cancer, Frederick, MD, USA, 10/10/12 16
  17. 17. Sample & Assay Technologies Gene expression: Serotonin metabolism Cholangiocarcinomas upregulate TPH and downregulate MAOA Tryptophan TPH1/2 DDC Serotonin MAOA/B Serotonin metabolites Metabolism and cancer, Frederick, MD, USA, 11/14/12 17
  18. 18. Sample & Assay Technologies Gene expression: Serotonin metabolism Increased serotonin secretion results in tumor growth Tryptophan TPH1/2 DDC Serotonin MAOA/B Serotonin metabolites Metabolism and cancer, Frederick, MD, USA, 11/14/12 18
  19. 19. Sample & Assay Technologies Metabolism & cancer experimental design QIAGEN offers a variety of products for metabolism & cancer Mutational analysis qPCR detection Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Metabolism and cancer, Frederick, MD, USA, 11/14/12 19
  20. 20. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Metabolism and cancer, Frederick, MD, USA, 11/14/12 20
  21. 21. Sample & Assay Technologies miRNA products from QIAGEN miRNeasy Isolation Target identification miRNA studies 3’ UTR reporters miRNA Targets Arrays Expression miScript miRNA PCR Array & Assays Function miRNA mimics & inhibitors Metabolism and cancer, Frederick, MD, USA, 11/14/12 21
  22. 22. Sample & Assay Technologies miRNA expression: Glycolysis Background: Hexokinase 2 has been linked to tumor progression. HK2 upregulation is part of the Warburg effect (aerobic glycolysis) Is HK2 epigenetically regulated? Methods: Comparison of normal human keratinocytes and head and neck squamous cell carcinoma (HNSCC) cells. Predict miRNAs that may regulate HK1 (miR-138) and HK2 (miR-143) via bioinformatic algorithms (TargetScan and PicTar) Metabolism and cancer, Frederick, MD, USA, 11/14/12 22
  23. 23. Sample & Assay Technologies miRNA expression: Glycolysis miR-143 downregulation leads to HK2 upregulation and increased tumor proliferation Metabolism and cancer, Frederick, MD, USA, 11/14/12 23
  24. 24. Sample & Assay Technologies miRNA expression: Glycolysis Background: Hexokinase 2 has been linked to tumor progression. HK2 upregulation is part of the Warburg effect (aerobic glycolysis) Is HK2 epigenetically regulated? Conclusion: miR-143 is a part of the Warburg effect, dysregulating glucose metabolism during tumorigenesis. Metabolism and cancer, Frederick, MD, USA, 11/14/12 24
  25. 25. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Metabolism and cancer, Frederick, MD, USA, 11/14/12 25
  26. 26. Sample & Assay Technologies DNA methylation: Glycolysis Background: Normal brain expresses HK1, but gliomas often overexpress HK2. Contributes to Warburg effect Embryos also express HK2; are gliomas reverting to an earlier developmental stage? Metabolism and cancer, Frederick, MD, USA, 11/14/12 26
  27. 27. Sample & Assay Technologies DNA methylation: Glycolysis HK2 downregulation correlates with promoter hypermethylation. Metabolism and cancer, Frederick, MD, USA, 11/14/12 27
  28. 28. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Metabolism and cancer, Frederick, MD, USA, 11/14/12 28
  29. 29. Sample & Assay Technologies Histone modifications: TCA cycle Oligodendromas often have the IDH1 R132H Mutation, increasing 2-HG levels Background: Increased 2-HG levels globally affect histone modifications Decreasing gene transcription Methods: Gene expression microarray of grade II-III oligodendroma samples. Identified IDH1/2 mutations Overexpress normal and mutant IDH1/2 in 3T3-L1 preadipocytes Examine histone modifications via ChIP during adipocyte differentiation Metabolism and cancer, Frederick, MD, USA, 11/14/12 29
  30. 30. Sample & Assay Technologies Histone modifications: TCA cycle Histone modifications reduce expression of key genes involved in adipocyte differentiation. Metabolism and cancer, Frederick, MD, USA, 11/14/12 30
  31. 31. Sample & Assay Technologies Metabolism & cancer experimental design QIAGEN offers a variety of products for metabolism & cancer Mutational analysis qPCR detection Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Metabolism and cancer, Frederick, MD, USA, 11/14/12 31
  32. 32. Sample & Assay Technologies Reporter assay overview Metabolism and cancer, Frederick, MD, USA, 11/14/12 32
  33. 33. Sample & Assay Technologies Reporter assay: Warburg effect Background: Hypoxic tumors release high levels of lactate Due to overexpression of the transporter MCT1 High lactate levels activate HIF-1 Metabolism and cancer, Frederick, MD, USA, 11/14/12 33
  34. 34. Sample & Assay Technologies Poor cancer prognosis: High lactate and high HIF1A Are Lactate levels and HIF1A inter-related? Metabolism and cancer, Frederick, MD, USA, 11/14/12 34
  35. 35. Sample & Assay Technologies Lactate/HIF1A conclusion Proposed mechanism for oncogenic angiogenesis Metabolism and cancer, Frederick, MD, USA, 11/14/12 35
  36. 36. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Metabolism and cancer, Frederick, MD, USA, 11/14/12 36
  37. 37. Sample & Assay Technologies shRNA: Warburg effect Background: The Warburg effect upregulates HIF1A activity PDK1 inhibits pyruvate formation, enhancing the Warburg effect PDK1 is regulated by HIF1A, forming a feed-forward loop How does this PDK1 mechanism contribute to tumorigenesis? Metabolism and cancer, Frederick, MD, USA, 11/14/12 37
  38. 38. Sample & Assay Technologies shRNA: Warburg effect PDK1 Epigenetic s IDH 2-HG Metabolism and cancer, Frederick, MD, USA, 11/14/12 38
  39. 39. Sample & Assay Technologies shRNA: Warburg effect PDK1 inhibition decreases tumorigenesis Metabolism and cancer, Frederick, MD, USA, 11/14/12 39
  40. 40. Sample & Assay Technologies Conclusions Many metabolic pathways important for oncogenesis Glycolysis/warburg effect TCA cycle Amino acid/serotonin metabolism Lipid metabolism/oxidative phosphorylation QIAGEN offers many methods to study these cellular processes Mutational analysis qBiomarker Somatic Mutation PCR Arrays & Assays qBiomarker Copy Number PCR Arrays & Assays Gene expression RT2 Profiler PCR Arrays & Assays Epigenetics miScript miRNA PCR System EpiTect Methyl II PCR Arrays & Assays EpiTect ChIP qPCR Arrays & Assays Functional studies Cignal Reporter Assays SureSilencing shRNA Plasmid Flexitube/Flexiplate siRNA, GeneSolution, Premix, AllStar Control Metabolism and cancer, Frederick, MD, USA, 11/14/12 40
  41. 41. Sample & Assay Technologies Thank you for attending! Are you ready to try? Please contact us today US and Canada Customers: Contact Technical Support: 9 AM-6 PM M – F EST Call: 1-888-503-3187 Email: support@SABiosciences.com International Customers: Call: 1-800-362-7737; 800-362-7737 Email: sabio@qiagen.com EU Customers: Email: sab.eu@qiagen.com Metabolism and cancer, Frederick, MD, USA, 11/14/12 41

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