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Tumor Biology: Cellular and Molecular Aspects of the Transformed Cell Growth Factors, Receptors, and Signal Transduction II Rebecca Riggins 202.687.7451 [email protected] Overview o Intracellular signaling “downstream” of receptors - key pathways: Ras/MAPK, Src, PI3K o Intracellular signaling defects in cancer o Targeted therapies to intracellular signaling molecules o TNF and TRAIL Brief Review – Growth Factors and Receptors o most growth factors are secreted o receptors are transmembrane proteins o 3 major features: o extracellular domain o transmembrane region o intracellular domain o where, when, and how they are expressed determines their biological function Brief Review – Receptor Activity o intracellular, or catalytic, domain has kinase activity o kinases add phosphate groups to (phosphorylate) specific amino acids ATP-binding Phospho-transferase ATP P ADP Amino Acid P Consequences of RTK activation GROWTH FACTOR PIP3 PIP3 RTK RAS SOS Grb2 P RAS P P PDK1 Akt p85 p110 PI3K Raf P MEK P ERK P BAD P P NF-ĸB PROLIFERATION CELL SURVIVAL FKHR P P P MDM2 GSK3 p70S6K PROTEIN SYNTHESIS . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). Intracellular Signaling o begins with autophosphorylated or transphosphorylated amino acids on the receptor o Phosphorylation recruits other proteins to the receptor o Amino acids surrounding the phosphorylation site determine which proteins are bound… 4 major protein interaction domains: SH2, PTB, SH3, PH Kinase Domain Membrane -Tyr P -Tyr P Basics of Amino Acid Structure Basics of Protein Structure o Primary Structure = “beads on a string” o Quaternary Structure = specific folding creates domains, or “units” of the protein SH2 domains o SH2 = src homology 2 o was first identified as a 100 amino acid region of homology (“sameness”) in the src tyrosine kinase o specifically recognizes phosphorylated Tyrosine o 2 classes of SH2 domain-containing proteins… - have enzymatic activity (like Src) - don’t have enzymatic activity o Those with no enzymatic activity bind other proteins to the receptor…adaptor molecules SH2 domain specificity o specificity is determined by the amino acids C-terminal to the phospho-Tyr o in most cases, it is the amino acid at position +3 that determines specificity Hydrophobic amino acid Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997 PTB domains o phosphotyrosine binding domains recognize amino acids N-terminal to the phospho-Tyr o PTB-containing proteins also participate in hormone signaling any amino acid Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997 SH3 domains o src homology 3 domains recognize amino acid sequences rich in Proline (Pro-rich) o Is a more general protein-protein interaction motif… many cytoskeletal proteins contain it any amino acid Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997 PH domains o pleckstrin homology domains recognize phospholipids (components of the plasma membrane) Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997 Protein-Protein Interactions and Receptors o Proteins with many different interaction domains can bind to growth factor receptor family members o Protein-protein interactions connect receptors to their intracellular signaling networks Figure 6.9 The Biology of Cancer (© Garland Science 2007) Receptors bind other Kinases, and Adaptor Proteins Figure 6.10a The Biology of Cancer (© Garland Science 2007) EGFR and the Ras Pathway EGF EGFR RAS SOS Grb2 P P Raf P MEK P ERK Grb2: adaptor protein that binds to phosphorylated Tyr on EGFR using its SH2 domain PROLIFERATION CELL SURVIVAL . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). Grb2 has multiple protein interaction domains Cell. 2004 Jan 23;116(2):191-203. EGFR and the Ras pathway, cont’d. EGF EGFR RAS SOS Grb2 P P Raf P MEK SOS binds to the Grb2 SH3 domain P ERK PROLIFERATION CELL SURVIVAL . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). SOS activates Ras o SOS is an exchange factor (it exchanges one nucleotide for another) Proline-rich DH PH Dbl-Homology CDC25 Catalytic Domain Membranetargeting SH3-Binding Grb2 Rho GTPases (Rac) EGFR A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005). What is Ras? o Ras is an oncogene o Ras is a small GTP-binding protein…it binds guanine triphosphate o Ras bound to GTP is active…Ras bound to GDP is inactive o Ras mutation is implicated in many kinds of cancer… A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005). Ras Mutations display Tumor Specificity Pancreatic Carcinoma K-Ras codon 12 (GGTgly) >GTTval Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser Bladder Carcinoma H-Ras codon 12 (GGCgly) >GTCval Melanoma N-Ras codon 61 (CAAgln)>CGAarg A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005). How is Ras activated? Figure 5.30 The Biology of Cancer (© Garland Science 2007) What does active Ras do? Autocrine growth factor signaling Figure 5.32a The Biology of Cancer (© Garland Science 2007) What else does active Ras do? EGF EGFR RAS SOS Grb2 P P Raf P MEK P ERK Ras activates Raf and the Erk/MAPK pathway PROLIFERATION CELL SURVIVAL . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). The Erk/MAPK pathway o Erk = extracellular signal regulated kinase o MAPK = mitogen activated protein kinase o MAPK promotes cell growth and survival by phosphorylating other proteins o An immediate consequence of MAPK activation is transcription (DNA → RNA) o MAPK activation is a major event following EGF stimulation… Nature Reviews Cancer 4, 937-947 (2004) Inhibitors of the MAPK Pathway Nature Reviews Cancer 4, 937-947 (2004) Mechanisms of Ras/MAPK inhibitors o inhibit Ras binding to the plasma membrane o inhibit Raf o inhibit MEK o some of these have entered clinical studies… EGF RAS SOS Grb2 EGFR P P Raf P MEK P ERK . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). EGFR and the PI3K pathway GROWTH FACTOR PIP3 PIP3 RTK P P P PDK1 Akt p85 p110 PI3K P BAD P P NF-ĸB FKHR P P MDM2 GSK3 CELL SURVIVAL . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). What is PI3K? Kinase Domain o PI3K = phosphatidyl inositol 3-kinase o phosphatidyl inositol is a lipid, part of the plasma membrane o so, PI3K phosphorylates lipids (fats) instead of other proteins o There are 2 subunits of PI3K… -Tyr P p85 p110 -Tyr P p85 = regulatory subunit p85 has an SH2 domain that binds phospho-Tyrosine on the EGFR PI3K p110 = catalytic subunit that phosphorylates lipids PI3K Target(s) o PI3K phosphorylates PIP2 to make PIP3 o PIP3 is now a binding site for Akt… Figure 6.19a The Biology of Cancer (© Garland Science 2007) Akt – the real master regulator o Akt is a Serine/Threonine kinase o Akt has targets in the cytoplasm as well as the nucleus o Akt inhibits the cell cycle inhibitors o inhibitor + inhibitor = GROWTH, and SURVIVAL GROWTH FACTOR PIP3 RTK P P P Akt p85 p110 PI3K P BAD P P NF-ĸB FKHR P P MDM2 GSK3 S G2 CELL SURVIVAL M G1 Cyclins, Cyclin-dependent kinases, and inhibitors . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005). Table 6.3 The Biology of Cancer (© Garland Science 2007) PI3K/Akt Defects in Cancer RTK GF Cancer Syndromes p PIP3 PI3-K Akt1/2 Lipid Kinase GI, Br, Ov Ser/Thr Kinase PANC Hamartin Tuberin (Tuberous Sclerosis TSC1 TSC2 Complex) (Ras-homology enriched in brain) Inhibitors to PI3K and/or Akt are being developed for patient use RheB (Target of rapamycin) S6K mTOR 4EBP-1 Protein synthesis Cell growth/size/survival Kovich & Cohen (2004) Dematology Online Journal 10: 3. Perelman (2004) Dematology Online Journal 10: 17. pp60 c-Src o “normal,” cellular Src is another protooncogene o viral Src (v-src) is the transforming gene of the avian Rous sarcoma virus o Src is a tyrosine kinase, but NOT a receptor o Cooperation/synergy with the EGFR in promoting proliferation and tumorigenesis in breast cancer cells Kinase Domain Phosphorylation of transcription factors -Tyr P Src -Tyr P Cell Growth MAPK and PI3K pathways c-Src phosphorylates many cellular proteins Isolate protein and load onto poly-acrylamide gel; detect phospho-Tyrosine with specific antibodies Figure 5.7a The Biology of Cancer (© Garland Science 2007) c-Src and Cancer o 30-70% of breast cancers overexpress Src, show elevated activity…many of these also overexpress EGFR o Other cancers Src may contribute to are: colon, lung, skin, endometrial, head/neck o Src is usually not mutated…how is it activated? Regulation of Src Activity Kinase SH3 “Inactive” N Kinase domain has no access to target proteins SH2 pY527 C “Active” N SH3 X SH2 Y Viral Src has lost this Tyrosine Kinase Kinase (p)Y527 C Kinase domain is now accessible How does Src become activated? o Src can bind to EGFR or PDGFR, and the active configuration is stabilized o However, are other proteins that can bind to and activate Src besides RTKs… “Active” N SH3 X SH2 Kinase Kinase (p)Y527 C Y Adaptor proteins with the right binding sites for SH2 and SH3 domains could activate Src The adaptor protein Cas can activate Src “Active” N SH3 RP640LPSPP Kinase SH2 Kinase (p)Y527 C pY668DYV Cas o Cas is important for cell proliferation, cell migration, and transformation by oncogenes (including viral Src) Cas, Src, and Breast Cancer o Cas, like Src, can also be overexpressed in breast cancer o Cas and Src bind to each other in breast cancer cells o Cas and Src are localized to the same areas of breast cancer cells o Cas overexpression in breast cancer is associated with resistance to a particular kind of drug (Tamoxifen)…this involves Src activation Cas Src Cas + Src Inhibitors of Src in Cancer Therapy o Inhibit protein-protein interactions (like those with Cas) o Inhibit kinase activity (preclinical/phase I trials) o Inhibit protein stability (accelerate Src degradation) o These will likely be used in combination with other chemotherapy drugs, EGFR inhibitors, etc. Adhesion Receptors Integrins Figure 5.28b The Biology of Cancer (© Garland Science 2007) Tyrosine Kinases and Adhesion Receptors: Overlap in Cytoplasmic Signaling Figure 6.24b The Biology of Cancer (© Garland Science 2007) G protein-coupled receptors: More overlap Figure 6.28 The Biology of Cancer (© Garland Science 2007) Tumor Necrosis Factor (TNF) Review o in some cells, TNF is mitogenic, but in most it promotes cell death, or apoptosis o this growth factor is not soluble, but is inserted into the plasma membrane o TNF receptors have no catalytic domain…rely on intracellular proteins to signal Intracrine Juxtacrine TNF family of growth factors Schulze-Osthoff, Trends Cell Biol. 1994 Dec;4(12):421-426. TNFR Signaling to Death DD = death domain, another protein interaction motif DD and DED form dimers DED = death effector domain Sci STKE. 2004 Jun 22;2004(239) CELL DEATH TNFR Signaling to Survival Other proteins CELL SURVIVAL TNF Signaling and cancer therapy: Will this work? Sci STKE. 2004 Jun 22;2004(239) TRAIL Signaling to Survival and Death TRAIL = TNF-related apoptosis-inducing ligand