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Sample & Assay Technologies
Metabolism and cancer
Sample & Assay Technologies Biology-focused solutions for pathway analysis
Metabolism and cancer, Frederick, MD, USA, 11/14/12 2
• SABiosciences is now a Company
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 cellsThis 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
Sample & Assay Technologies Metabolic pathway overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 4
Levine, A.J. (2010) Science 330, 1340.
Warburg effect
Sample & Assay Technologies Metabolic regulators overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 5
Many oncogenes and tumor suppressor genes regulate metabolism
Munoz-Pinedo, C. (2012) Cell Death & Dis. 3, e248.
Sample & Assay Technologies Metabolism & cancer experimental design
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
QIAGEN offers a variety of products for metabolism & cancer
Sample & Assay Technologies Somatic mutation analysis: Overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 7
Hydrolysis probe + ARMS (Amplification Refractory Mutation System)
F Q
Common Region
F Q
Sample & Assay Technologies Key metabolic mutations in cancer
Metabolism and cancer, Frederick, MD, USA, 11/14/12 8
IDH 2-HG
Epigenetics
Oncogenes
TS Genes
Modified from: Deberardinis, R.J. (2008) Genet. Med. 10, 767.
Sample & Assay Technologies Somatic mutation: TCA cycle
Metabolism and cancer, Frederick, MD, USA, 11/14/12 9
IDH1 and IDH2 are currently hot topics
Sample & Assay Technologies Somatic mutation: TCA cycle
Metabolism and cancer, Frederick, MD, USA, 11/14/12 10
IDH1 R132H shows altered metabolic activity.
Reitman, Z.J. (2010) JNCI. 102, 932.
Sample & Assay Technologies Copy Number Variation (CNA/CNV) overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 11
Discovery
• Array CGH• SNP Chips• NGS
Validation
• FISH• qPCR
Diagnostic test
Sample & Assay Technologies CNV: Cancer & metabolism
Metabolism and cancer, Frederick, MD, USA, 11/14/12 12
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.
Sample & Assay Technologies CNV: Cancer & metabolism
Metabolism and cancer, Frederick, MD, USA, 11/14/12 13
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
Sample & Assay Technologies Metabolism & cancer experimental design
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
QIAGEN offers a variety of products for metabolism & cancer
Sample & Assay Technologies Principles of qRT-PCR: Overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 15
Ct values: Threshold cycle
Real-time PCR� Amplify and simultaneously quantify target DNAReverse transcription real-time PCR� Amplify and simultaneously quantify mRNA
Sample & Assay Technologies Gene expression: Serotonin metabolism
Metabolism and cancer, Frederick, MD, USA, 10/10/12 16
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 reportedMethods: Grow multiple cholangiosarcoma cell lines� Examine serotonin metabolism via direct measurement and gene expression of
metabolic pathways� Confirm hypothesis with nude mice
Sample & Assay Technologies Gene expression: Serotonin metabolism
Metabolism and cancer, Frederick, MD, USA, 11/14/12 17
Cholangiocarcinomas upregulate TPH and downregulate MAOA
Tryptophan
Serotonin
TPH1/2
DDC
MAOA/B
Serotonin metabolites
Sample & Assay Technologies Gene expression: Serotonin metabolism
Metabolism and cancer, Frederick, MD, USA, 11/14/12 18
Tryptophan
Serotonin
TPH1/2
DDC
MAOA/B
Serotonin metabolites
Increased serotonin secretion results in tumor growth
Sample & Assay Technologies Metabolism & cancer experimental design
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
QIAGEN offers a variety of products for metabolism & cancer
Sample & Assay Technologies Epigenetics overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 20
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
Sample & Assay Technologies miRNA products from QIAGEN
Metabolism and cancer, Frederick, MD, USA, 11/14/12 21
3’ UTR reportersmiRNA Targets Arrays
Target identification Expression
Isolation
Function
miRNA mimics & inhibitors
miScript miRNA PCR Array & Assays
miRNeasy
miRNAstudies
Sample & Assay Technologies miRNA expression: Glycolysis
Metabolism and cancer, Frederick, MD, USA, 11/14/12 22
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)
Sample & Assay Technologies miRNA expression: Glycolysis
Metabolism and cancer, Frederick, MD, USA, 11/14/12 23
miR-143 downregulation leads to HK2 upregulation and increased tumor proliferation
Sample & Assay Technologies miRNA expression: Glycolysis
Metabolism and cancer, Frederick, MD, USA, 11/14/12 24
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.
Sample & Assay Technologies Epigenetics overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 25
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
Sample & Assay Technologies DNA methylation: Glycolysis
Metabolism and cancer, Frederick, MD, USA, 11/14/12 26
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?
Sample & Assay Technologies DNA methylation: Glycolysis
Metabolism and cancer, Frederick, MD, USA, 11/14/12 27
HK2 downregulation correlates with promoter hypermethylation.
Sample & Assay Technologies Epigenetics overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 28
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
Sample & Assay Technologies Histone modifications: TCA cycle
Metabolism and cancer, Frederick, MD, USA, 11/14/12 29
Oligodendromas often have the IDH1 R132H Mutation, increasing 2-HG levels
Background: Increased 2-HG levels globally affect histone modifications� Decreasing gene transcriptionMethods: 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
Sample & Assay Technologies Histone modifications: TCA cycle
Metabolism and cancer, Frederick, MD, USA, 11/14/12 30
Histone modifications reduce expression of key genes involved in adipocyte differentiation.
Sample & Assay Technologies Metabolism & cancer experimental design
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
QIAGEN offers a variety of products for metabolism & cancer
Sample & Assay Technologies Reporter assay overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 32
Sample & Assay Technologies Reporter assay: Warburg effect
Metabolism and cancer, Frederick, MD, USA, 11/14/12 33
Background: Hypoxic tumors release high levels of lactate� Due to overexpression of the transporter MCT1� High lactate levels activate HIF-1
Sample & Assay Technologies Poor cancer prognosis: High lactate and high HIF1A
Metabolism and cancer, Frederick, MD, USA, 11/14/12 34
Are Lactate levels and HIF1A inter-related?
Sample & Assay Technologies Lactate/HIF1A conclusion
Metabolism and cancer, Frederick, MD, USA, 11/14/12 35
Proposed mechanism for oncogenic angiogenesis
Sample & Assay Technologies Epigenetics overview
Metabolism and cancer, Frederick, MD, USA, 11/14/12 36
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
Sample & Assay Technologies shRNA: Warburg effect
Metabolism and cancer, Frederick, MD, USA, 11/14/12 37
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?
Sample & Assay Technologies shRNA: Warburg effect
Metabolism and cancer, Frederick, MD, USA, 11/14/12 38
IDH 2-HG
Epigenetics
PDK1
Sample & Assay Technologies shRNA: Warburg effect
Metabolism and cancer, Frederick, MD, USA, 11/14/12 39
PDK1 inhibition decreases tumorigenesis
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
ControlMetabolism and cancer, Frederick, MD, USA, 11/14/12 40
Sample & Assay Technologies Thank you for attending!
Metabolism and cancer, Frederick, MD, USA, 11/14/12 41
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