2. Sample & Assay Technologies
Biology-focused solutions for pathway analysis
• SABiosciences is now a
Company
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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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
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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
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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
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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
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Q
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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
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9. Sample & Assay Technologies
Somatic mutation: TCA cycle
IDH1 and IDH2 are currently hot topics
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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27. Sample & Assay Technologies
DNA methylation: Glycolysis
HK2 downregulation correlates with promoter hypermethylation.
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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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
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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
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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
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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
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32. Sample & Assay Technologies
Reporter assay overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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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
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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
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35. Sample & Assay Technologies
Lactate/HIF1A conclusion
Proposed mechanism for oncogenic angiogenesis
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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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
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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
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38. Sample & Assay Technologies
shRNA: Warburg effect
PDK1
Epigenetic
s
IDH
2-HG
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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39. Sample & Assay Technologies
shRNA: Warburg effect
PDK1 inhibition decreases tumorigenesis
Metabolism and cancer, Frederick, MD, USA, 11/14/12
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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
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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
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