Surp09 Signaling


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2009 presentation to summer undergraduates at Science Park participating in summer research program. Presentation on "signal transduction and cancer"

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Surp09 Signaling

  1. 1. Signal Transduction and Cancer SURP Program July 7, 2009
  2. 2. Outline of Today’s Lecture 8 Signal transduction overview 8 Why important in cancer? 8 Examples of important pathways 8 How signaling is studied in the lab 8 Signal transduction pathways as therapeutic targets 2
  3. 3. What is signal transduction? 8 The process by which extracellular signals are transmitted across the cell membrane and converted into a cellular response (e.g. gene expression, apoptosis…) 8 “the process by which a cell converts a signal or stimulus into another”. ( 8 “How cells respond to environmental stimuli” 3
  4. 4. Typical Components of Signaling Pathways Receptor Target Ligand Effector/ (Stimulus) Amplification Adaptor of signal proteins Second Messengers Biological Response 4
  5. 5. Types of Signals 8 Proteins: – Cytokines (e.g. IL-1) – Growth factors (e.g. EGF, TGFβ) 8 Nutritional factors: – Sugars (glucose) – Amino Acids – Lipids – Vitamins 8 Hormones (e.g. adrenaline, estrogen, insulin) 8 Other: – Stress, heat, pH, radiation, etc 5
  6. 6. Signals can act in a cell-type specific manner Mesenchymal cells oting th prom (e.g. fibroblasts) Grow TGFβ Grow th inhibi ting Epithelial cells Endothelial cells Hemopoietic cells Neural cells 8 Important to note specific context when evaluating signaling literature! 6
  7. 7. Receptors 8 Growth factors signal via receptor tyrosine kinases (RTKs/RPTKs). 8 Transmembrane proteins, containing an extracellular domain of several hundred AAs, short alpha helix structured hydrophobic domain spanning the membrane, and an intracellular catalytic domain that is highly conserved among family members. 8 Ligand binding induces a change in conformation of receptors, allowing dimerization and transphosphorylation of Tyr residues. 8 Signaling intermediates are recruited to the phosphorylated (“active”) receptor to propagate the signal. 7
  8. 8. Receptor Activation Schematic Ligand Molecular Biology of the Cell, Alberts et al. Ch 3 8
  9. 9. Phosphorylation and dephosphorylation regulate the activity of many proteins 8 Kinases: Enzymes that catalyze the removal of a phosphate group from ATP which is transferred to serine, threonine or tyrosine residues in proteins. – 2 classes: Ser/Thr and Tyr kinases 8 Phosphatases: Enzymes which catalyze the removal of phosphate groups from proteins Molecular Biology of the Cell, Alberts et al. Ch 3 9
  10. 10. Effectors 8 Anchor proteins localize protein kinases and phosphatases in particular places in the cell – Increases efficiency and specificity of responses 8 Protein-protein complexes form via interactions between specific protein domains (e.g. SH2 domains which recognize phospho-tyrosines) 10
  11. 11. Biological outcomes 8 Changes in gene expression 8 Regulate translation 8 Regulate the cell cycle 8 Migrate 8 Reorganize the cytoskeleton 8 Initiate angiogenesis etc 11
  12. 12. Cancer and growth control 8 Normal cells respond to their environment to maintain a balance between proliferation, differentiation and cell death. 8 Cancer cells have defects in growth control, leading to… Hahn and Weinberg Cell 2000 12
  13. 13. Why study signal transduction in cancer? Nearly all proto-oncogenes are members of signal transduction pathways involved in regulating cell proliferation, differentiation or apoptosis “Signal transduction” in title/abstract QuickTimeª and a decompressor are needed to see this picture. Total: 57037 (as of 7/6/09) 13
  14. 14. What goes wrong in cancer? 8 Tumors cells inappropriately secrete growth factors that activate cognate receptors on their cell surface - allows signaling independent of “true Example: Renal cell cell-cell carcinoma cells have an communication” autocrine TGFα - EGFR autocrine loop 14
  15. 15. What goes wrong in cancer (2) 8 Upregulation of growth factor receptors 8 Deletions in receptor domains causing defects in ligand binding, or causing constitutive dimerization. 8 Normal cells contain 20,000-200,000 copies of EGFR; cancers cells can contain >1,000,000! – Greatly increased upstream mitogenic signaling. Classic example: Breast cancer cells can overexpress HER2 (an EGFR family member) 15
  16. 16. What goes wrong in cancer? (3) 8 Increase in signal transduction – Loss of negative regulators (e.g. mutation or epigenetic silencing of PTEN in endometrial carcinoma) – Defects in protein turnover – Defects in protein localization (e.g. sequestration of p27 in to the cytoplasm in RCC) – Novel oncogenes from chromosomal translocations that result in constitutive activity of a kinase (e.g. Bcr- Abl) 8 Increase in activation of transcription – Constitutive activation of transcription factors (e.g. STAT3) 16
  17. 17. Signal transduction pathways are targets of carcinogens 8 Carcinogen (e.g. DMBA) causes an activating mutation in codon 61 of H-ras. 8 Tumor promoters like TPA promote clonal expansion of initiated cells. Multiple signaling pathways targeted by TPA including PI3K-Akt, and protein kinase C. 17
  18. 18. Overview of most important signal transduction pathways in cancer 8 PI3K-Akt-mTOR SURVIVAL C (regulate protein R translation, cell O growth, autophagy) S S 8 Ras-MAPK PROLIFERATION T 8 JAK-STAT PROLIFERATION A (anti-apoptosis, L K immune response) 8 NFκB INFLAMMATION 8 WNT-β-Catenin DIFFERENTIATION 18
  19. 19. How signal transduction is studied 8 Cell culture studies: – Use of inhibitors (e.g. rapamycin, LY294002) – Phospho-specific antibodies – If no antibodies available, can immunoprecipitate and western blot with broad phospho-tyr or phospho- ser/thr antibodies – In vitro kinase reactions: determine whether a specific kinase can phosphorylate a given substrate in a test tube. – Other enzyme assays which measure products 19
  20. 20. How signal transduction is studied (2) 8 In vivo studies: – Phospho-specific antibodies for immunohistochemistry – Commercially available/custom tissue microarrays to look at many samples at once – Transgenic /knockout mouse models 20
  21. 21. Signal tranduction as a therapeutic target 8 Traditional cytotoxic chemotherapy has problems: – Non-specific, unpleasant side effects --> kills proliferating cells, even normal ones such as hair follicles, GI epithelial cells – Cells develop resistance mechanisms (e.g. upregulate proteins that pump drugs out of the cells so they can no longer accumulate) 8 Rationally designed targeted therapies that interfere with signaling the current rage in cancer therapeutics research. – Ideal target = a protein not involved in normal cellular function (Bcr-Abl example in next few slides) 21
  22. 22. Determining a good drug target 8 Determine what pathways are active in your tumor type 8 Determine whether any of these pathways are critical for tumor survival – “Oncogene addiction” phenomenon: Despite the fact that tumors contain multiple genetic and epigenetic defects, their growth/survival can often be impaired by inactivation of a single oncogene (the “Achilles Heel”)! 8 Financially worthwhile - enough of a target market (large enough prevalence) 8 2 examples: Bcr-Abl and HER2/neu 22
  23. 23. Bcr-Abl in CML 8 Chromosomal translocation results in a constitutively active tyrosine kinase. 8 Found in 95% of CML patients 8 Signals via a multitude of growth-promoting pathways (PI3K, JAK- STAT, Myc, NFκB) 23
  24. 24. Gleevec 8 Gleevec is a small molecule designed to bind to the ATP cleft and inhibit the tyrosine kinase activity of Bcr-Abl at low conc’s (40nM). – Later found to also inhibit PDGFR and c-Kit QuickTimeª and a 8 Successfully used so far: decompressor are needed to see this picture. – 98% complete response rate in chronic phase CML (NEJM, 2001) – Reduce total tumor cells from 1010/1012 to 106 (minimal residual disease) – Now is the standard of care! 24
  25. 25. HER2 as a target in breast cancer 8 Some breast cancers (~25%) overexpress the HER2 gene, a growth factor receptor in the EGFR family. 8 These tumors have a worse prognosis than non HER2- overexpressing tumors. 8 Antibodies such as Herceptin (trastuzumab) have been developed to block the aberrant mitogenic signaling. 8 Good results are seen in synergy with standard therapies (chemo and radiation) 25
  26. 26. Sample of what pharma has done 26
  27. 27. Take home points 8 Signal transduction is the mechanism by which cells communicate. 8 Signal transduction networks are often bizarre in cancer and result in an imbalance between cell death and cell survival. 8 Multiple mechanisms are employed by cancer cells to subvert normal regulation of proteins involved in signaling, including gene amplification, mutations, chromosomal translocations and epigenetic silencing of tumor suppressors. 8 Rationally designed signaling inhibition is a relevant therapeutic approach. 27
  28. 28. Additional Resources 8 Cell Signaling Technologies website - (lots of signaling diagrams) and (information about kinases and their substrates) 8 Science Signal Transduction Knowledge Environment - 8 Calbiochem interactive pathways - activepathways.htm Questions? 28