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Possible Link between Cancer Multidrug Resistance and Epigenetics
Erin Wildeman, Marcos Pires, PhD
Lehigh University Department of Chemistry, Bethlehem, PA 18015
Rationale
Outline of Project:
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Cancer is a widespread disease, affecting over 1.5 million patients each year
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Treatment of most cancers is heavily reliant on chemotherapeutic drugs
Histones
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Most chemotherapeutic treatments often fail to eliminate 100% of the cancerous
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Histones are a four protein complex (Two of each, H2A, H2B, H3, and H4)
cells from a patient’s body
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DNA wraps around histone complex when condensing into its condensed chromosome structure
- Some cancer patients experience a relapse
1. Express the PHD3 domain of RBP2
2. Synthesize peptides to mimic histone tail
3. Measure binding affinity of PHD3 to different peptides
Current Status of Project:
- The relapse tumor is usually more aggressive than the original tumor
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Current Research
Background Continued
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Understanding why relapse tumors emerge more aggressively would allow cancer
PHD3 domain is expressing and has been successfully purified
- Amplified PHD3 from plasmid containing the RBP2 gene using PCR
- Cloned into pGEX-4T-1 vector, in frame with a GST tag
- Transformed into BL-21 cells
- Grew cells on ampicillin plates
- Overnight Growth at 37 °C, induced with IPTG
- Centrifuged, re-suspended cells in 1X PBS
- Lysed cells via sonication
- Purified protein on a glutathione column
SDS-PAGE from protein purification:
therapies to be created that are more effective, increasing survival rates among
cancer patients
H3 Tail
Background Information
Small populations of cells from the original tumor can be converted to “Drug
Tolerant Persisters (DTPs)” or cancer stem cells
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Epigenetic Factors
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Cancer Stem Cells
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Journal of Biological Chemistry, 9 April 2010, Volume 285, pages 11045-11050
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“Heritable phenotypes resulting from changes in chromosomes without alterations in the primary
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9 10 11 12 13
DNA sequence”
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Types of Epigenetic Factors:
- Histone side-chain modifications (Methylation, phosphorylation, acetylation)
Characteristics of Cancer Stem Cells:
- Nucleosome placement
- Aggressive
- DNA Methylation (cytosine)
- Resilient
- Self-renewal ability (tumor metastasis)
- Resistant to the chemotherapeutic drug originally used
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Epigenetic Modifications have been linked to several types of diseases including many types of
cancer
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Synthesis of Polypeptide:
Use solid state peptide synthesis to create a peptide that mimics the Histone 3 tail
“Recent research suggests that the coexistence of modifications at nearby sites
modulates the binding affinity of PHD fingers”
Multiple different histone 3 tails will be synthesized, with a combination of different
epigenetic modifications
- Will be researching what modifications occur most commonly with Lysine 4
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Normal cells have a fully methylated Lys on histone three (H3K4me3)
- In cancerous cells, H3K4 exists in the demethylated state (H3K4me2 or H3K4me1)
Expression Compared to
Histone Modification:
Normal Tissue:
DTPs (cancer stem cells) constitute ~0.3% of the
original cell line population
Plate 1= untreated
Plate 2= treated for 9 days
Plate 3= treated every 3 days for 33 days
H3K4me1
Decreased, increased upon progression
Prostate
H3K4me2
Decreased, increased upon progression
Lung, Prostate, Kidney, Non Small Cell Lung
Carcinoma, Hepatocellular Carcinoma,
Breast, Pancreatic, Adenocarcinoma
H3K4me3
Decreased, increased upon progression
Prostate
Cell, 2 April 2010, Volume 141, Issue 1, Pages 69–80
RBP2
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The protein RBP2 was found to be over expressed in cancer stem cells
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RBP2 is a histone demethylase
- Specifically Lysine 4 on Histone 3 (H3K4)
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Believed to be the protein responsible for the conversion of normal cancer cells to
Associated Cancer
BBA-Reviews on Cancer, January 2011, Volume 1815, pages 75-89
PHD3 Domain
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The RBP2 protein has a catalytic domain as well as three PHD domains
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The PDH domain (Plant Homeodomain) is believed to be the “sensing domain”
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Research has proven that the PHD3 domain is critical for the function of RBP2
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RBP2 protein lacking the PHD3 domain was unable to interact with Histone 3
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RBP2 containing the PHD3 domain, when exposed to different H3 Lysine side chains, interacts
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Measure Binding Affinity of PHD3 Domain for Polypeptide
Future of Project:
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Little is currently known about how RBP2 functions
We hope to better understand the combination of epigenetic modifications on
histone 3 that code for effective binding of RBP2
Ultimate goal of project: Development an inhibitor for RBP2
- No pharmacological inhibitors have been developed yet for RBP2
- Inhibitor could improve outcome of cancer patients
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cancer stem cells
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In a knock-down experiment, the cells were unable to convert to the drug-tolerant
state
most strongly with H3K4
Cell, 2 April 2010, Volume 141, Issue 1, Pages 69–80
Nature, 11 June 2009, Volume 459, pages 847-851
References
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BBA-Reviews on Cancer, January 2011, Volume 1815, pages 75-89
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Cell, 2 April 2010, Volume 141, Issue 1, pages 69–80
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Journal of Biological Chemistry, 9 April 2010, Volume 285, pages 11045-11050
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Nature, 11 June 2009, Volume 459, pages 847-851
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Nature Biotechnology, 13 October 2010Volume 28,, pages 1057–1068
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Nature Chemical Biology, 28 February 2010, Volume 6, pages 283-290