Possible Link between Cancer Multidrug Resistance and Epigenetics Erin Wildeman, Marcos Pires, PhD Lehigh University Department of Chemistry, Bethlehem, PA Rationale Cancer is a widespread disease, affecting over 1.5 million patients each year Treatment of most cancers is heavily reliant on chemotherapeutic drugs Most chemotherapeutic treatments often fail to eliminate 100% of the cancerous cells from a patient’s body - Some cancer patients experience a relapse - The relapse tumor is usually more aggressive than the original tumor Understanding why relapse tumors emerge more aggressively would allow cancer therapies to be created that are more effective, increasing survival rates among cancer patients Background Continued Histones Histones are a four protein complex (Two of each, H2A, H2B, H3, and H4) DNA wraps around histone complex when condensing into its condensed chromosome structure H3 Tail Journal of Biological Chemistry, 9 April 2010, Volume 285, pages Epigenetic Factors “Heritable phenotypes resulting from changes in chromosomes without alterations in the primary DNA sequence” Types of Epigenetic Factors: - Histone side-chain modifications (Methylation, phosphorylation, acetylation) - Nucleosome placement - DNA Methylation (cytosine) Epigenetic Modifications have been linked to several types of diseases including many types of cancer Normal cells have a fully methylated Lys on histone three (H3K4me3) - In cancerous cells, H3K4 exists in the demethylated state (H3K4me2 or H3K4me1) BBA-Reviews on Cancer, January 2011, Volume 1815, pages PHD3 Domain The RBP2 protein has a catalytic domain as well as three PHD domains The PDH domain (Plant Homeodomain) is believed to be the “sensing domain” Research has proven that the PHD3 domain is critical for the function of RBP2 RBP2 protein lacking the PHD3 domain was unable to interact with Histone 3 RBP2 containing the PHD3 domain, when exposed to different H3 Lysine side chains, interacts most strongly with H3K4 Nature, 11 June 2009, Volume 459, pages Current Research Outline of Project: 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: 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: 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 Measure Binding Affinity of PHD3 Domain for Polypeptide Future of Project: 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 Background Information Cancer Stem Cells Small populations of cells from the original tumor can be converted to “Drug Tolerant Persisters (DTPs)” or cancer stem cells Characteristics of Cancer Stem Cells: - Aggressive - Resilient - Self-renewal ability (tumor metastasis) - Resistant to the chemotherapeutic drug originally used Cell, 2 April 2010, Volume 141, Issue 1, Pages 69–80 RBP2 The protein RBP2 was found to be over expressed in cancer stem cells RBP2 is a histone demethylase - Specifically Lysine 4 on Histone 3 (H3K4) Believed to be the protein responsible for the conversion of normal cancer cells to cancer stem cells In a knock-down experiment, the cells were unable to convert to the drug-tolerant state Cell, 2 April 2010, Volume 141, Issue 1, Pages 69–80 Plate 1= untreated Plate 2= treated for 9 days Plate 3= treated every 3 days for 33 days DTPs (cancer stem cells) constitute ~0.3% of the original cell line population Histone Modification: Expression Compared to Normal Tissue: Associated Cancer H3K4me1Decreased, increased upon progressionProstate H3K4me2Decreased, increased upon progression Lung, Prostate, Kidney, Non Small Cell Lung Carcinoma, Hepatocellular Carcinoma, Breast, Pancreatic, Adenocarcinoma H3K4me3Decreased, increased upon progressionProstate References BBA-Reviews on Cancer, January 2011, Volume 1815, pages Cell, 2 April 2010, Volume 141, Issue 1, pages 69–80 Journal of Biological Chemistry, 9 April 2010, Volume 285, pages Nature, 11 June 2009, Volume 459, pages Nature Biotechnology, 13 October 2010Volume 28,, pages 1057–1068 Nature Chemical Biology, 28 February 2010, Volume 6, pages