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Molecular and Cellular Aspects of Biosignaling Part II: Signaling, Cell Cycle, and Cancer March 2011 Yong Tae Kwon Cell Cycle and Cancer Why is cell cycle important? ~1.5 M new cancer patients in US ~0.5 M deaths in US ~13% of all deaths ~7.6 M deaths worldwide in 2007 Cell cycle Cell cycle checkpoint DNA repair Apoptosis Cancer All Connected!!! The Cell Cycle M-phase: mitosis and cytokenesis Interphase G1 (Gap1) S: DNA replication G2 G0: quiescent or senescent Homologous chromosomes Sister chromatids (replicated) G1 S G2 M kinetochore Cell cycle: G1-S-G2-M Microtubule kinesin G1 G2 Mitosis Prophase Anaphase mm pp mp + mp Metaphase Telophase Mitosis G1 The cell cycle controlled by inhibition for DNA integrity Cyclin-dependent kinase (CDK): key molecule cyclin: activator Kinase Phosphatase Ubiquitin-dependent proteolysis Core cell cycle machinery: CDK and Cyclin Cyclin-dependent kinase (CDK): key molecule Cyclin: activator Wee1: inhibitory kinase CAK: CDK-activating kinase CDC25: phosphatase APC-dependent ubiquitylation of cyclin Mitosis (M-phase) controlled by CDK and cyclin Cell cycle regulators destructed by ubiquitin-proteasome system M-phase M-cyclin APC E3 system G1/S-phase G1/S-cyclins SCF E3 system The ubiquitin system Phosphorylation vs. Ubiquitylation Enzymatic cascade in UPS 2 E1 ~50 E2 ~500 E3: major specificity ~2000 Substrates: many processes 1 Proteasome Monoubiquitylation: regulation vs. phosphorylation Polyubiquitylation: degradation The 26S Proteasome Universal proteolytic machinery 19S + 20S + 19S = 26S Valcade: proteasome inhibitors as anti-cancer drug How does extracellular signals induce cell growth? Growth factor and G1/S Entry into S phase Growth factor: autocrine or paracrine PDGF RTK Ras MAPK module Jun/Fos: transcription factors G1/S cyclins Receptor tyrosine kinase for growth factor PDGF Receptor tyrosine kinase Growth factors: EGF, PDGF etc RTK signaling and Ras GTPase MAPK cascade in RTS-Ras signaling RTK: kinase receptor Ras: GTPase MAPKKK: kinase MAPKK: kinase MAPK: kinase Transcription factors (e.g., c-Fos) Why multi-step in signaling? Signal amplified in number and duration Signal fine regulated Signal cross talks G1/S progression M-cyclin M-CDK CDK-cyclin combinations Wee1-like inhibitory kinase CAK: G1/S-CAK CDC25 SCF-dependent ubiquitylation of cyclin CDK/cyclin in G1/S Rb in G1/S progression Rb Retinoblastoma sensitivity protein CDK substrate E2F transcription factor Rb-E2F: inactive Rb-P + E2F G1/S protein expression Cell cycle checkpoint (G1/S) Cell cycle arrest when DNA damage found p53: transcription factor Cell cycle check point protein p53 phosphorylation p21: CDK inhibitor DNA repair Apoptosis G1/S checkpoint by p53 How is p53 activity induced by DNA damage? DNA damage and cell cycle arrest p53: transcription factor p53 transcribes p21. Cell cycle check point p53 induces E3 MDM2 transcription. MDM2 ubiquitylates p53. How is p53 activity induced by DNA damage? DNA damage and cell cycle arrest DNA damage DNA kinase p53 phosphorylation p21: CDK inhibitor DNA repair Apoptosis p53 mutation in >50% of cancers Apoptosis: death for survival Cell termination, homeostasis, development etc DNA damage p53: cell cycle arrest DNA repair vs. apoptosis Caspases: Cysteine protease cascade vs. blood coagulation Apoptosis Plasma membrane blebs Cell shrinkage Nucleus condensation (pyknosis) Endonucleases: degrade chromosome, ~170 bp Nucleus fragmentation Engulfed by phagocytes Apoptosis pathways Death receptor and mitochondrial apoptotic pathways Extracellular Intracellular endonucleases apoptosis Death receptor pathway (TNF) TNF (tumor necrosis factor): cytokine TNF receptor TRADD (TNFR-associated DD) FADD (Fas-associated DD) DD: death domain Caspase 8 Caspase cascade Apoptosis Mitochondrial apoptotic pathway Mitochondria: aspiration Pore: cytochrome c release Pore forming: Bax, Bak Pore facilitator: Bad, Bid Anti-pore: Bcl-2 Apoptosome Cytochrome c Apaf-1 Pro-caspase 9 Caspase 9 Apoptosis p53: cell cycle arrest and apoptosis The transcription factor p53 The ubiquitin ligase MDM2 p53 degradation DNA damage ATM, ATR kinases p53 phosphorylation and stabilization Cell cycle arrest (p21) and apoptosis Signaling, cell cycle, and cancer Cancer is the most serious threat to human health, and SOP students should not avoid problems in cancer Unregulated cell division Cell cycle checkpoint DNA repair Resistance to apoptosis Malignant cancer cells Induce angiogenesis Metastasis Oncogenesis Oncogenesis involves multiple events (e.g., p53 mutation + abnormal proliferation with Ras mutation) Cancer is a multi-stage disease Stage 0 Creation of cancer cells Stage I Restricted to one part of the body Stage II Locally advanced Stage III Locally advanced Stage IV Often metastasized, or spread to other organs or throughout the body. Sequential mutations involved and required Carcinogenesis Metastasis Spreading of cancer cells throughout the body. An environment where tumor cells can proliferate, invade surrounding tissues, be released into the circulation, invade a distant organ, establish their own blood supply (angiogenesis), and grow. Each of these steps as drug targets Cancer and angiogenesis VEGF (vascular endothelial cell growth factor) FGF (fibroblast growth factor) Blood vessel induction into tumor mass Nutrition and oxygen supply Cancer and angiogenesis Metastasis uPA (Urokinase plasminogen activator) Plasminogen - plasmin Procollagenases Collagenases Extracellular matrix breakdown Secreted proteases and metastasis Oncogene pathways Tumor suppressor pathways Protooncogene products that regulate cell division and apoptosis Activators of cell division Growth factors Growth factor receptors Tyrosine kinase (cytoplasmic) Adaptor proteins Ras GTP-binding proteins Kinases and kinase cofactors Phosphatases Transcription factors PDGF, FGF, etc PDGFR, EGFR, etc Src Grb-2 K-Ras, H-Ras, N-Ras Cyclins, CDK, CAK, MAPKs CDC25 Myc, Jun, Fos, E2F Activators of apoptotic resistance (survival) Bcl-2, Bcl-XL MDM2 XIAP Akt and Raf (MAPKKK) NFkB (transcription factor) What are mechanisms underlying oncogenesis? Let’s start from extracellular growth factors Entry into S phase Growth factor: autocrine or paracrine PDGF RTK Ras MAPK module Jun/Fos: transcription factors G1/S cyclins Receptor tyrosine kinase for growth factor PDGF Receptor tyrosine kinase Growth factors: EGF, PDGF etc RTK signaling and Ras GTPase MAPK cascade in RTS-Ras signaling RTK: kinase receptor Ras: GTPase MAPKKK: kinase MAPKK: kinase MAPK: kinase Transcription factors (e.g., c-Fos) Why multi-step in signaling? Signal amplified in number and duration Signal fine regulated Signal cross talks G1/S progression M-cyclin M-CDK CDK-cyclin combinations Wee1-like inhibitory kinase CAK: G1/S-CAK CDC25 SCF-dependent ubiquitylation of cyclin CDK/cyclin in G1/S Rb in G1/S progression Rb Retinoblastoma sensitivity protein CDK substrate E2F transcription factor Rb-E2F: inactive Rb-P + E2F G1/S protein expression Cell cycle checkpoint (G1/S) Cell cycle arrest when DNA damage found p53: transcription factor Cell cycle check point protein p53 phosphorylation p21: CDK inhibitor DNA repair Apoptosis G1/S checkpoint by p53 Broken cell cycle regulation is one critical factor But it is not sufficient… Rb Retinoblastoma protein CDK substrate E2F transcription factor Rb-E2F: inactive Rb-P + E2F G1/S protein expression p53 mutation in cancer ~50% of all cancer patients involve p53 mutation Constitutive activation of proliferation signals can be another factor p53 mutation: ~50% of all cancers Ras mutation: ~30% of all cancers On-off switch On-off switch broken Constitutively activated Mutations in RTK found in cancers Trastuzumab (Genentech): anti-HER2 antibody for HER2-overexpressingbreast cancers Cetuximab (ImClone): Antibody for EGF-binding domain Small molecule drugs How can virus induce cancer? Cervical cancer induced by HPV Human Papillomavirus Virus: DNA virus Skin, mucous membrane Benign skin warts, genital warts ~50% women infected Cervical cancer: 10,000 women (4,000 death) HPV induces uncontrolled proliferation: how? HPV targets p53 and pRb to induce cell proliferation and to inhibit apoptosis E6 p53 (degradation) E7 Rb (binding) Apoptosis Checkpoint G1/S HPV vaccine How about developing drugs that target E6-p53 interaction? How about proteasome inhibitor? The new vaccine to protect girls against HPV prompts debate about why it's necessary at such a young age. Cancer treatment Normal vs. cancer cells Proliferation Chemotherapy DNA mutagens Radiation therapy X-ray, ionizing radiations Drug Velcade, Herceptin Chemotherapy and radiation therapy DNA damage in proliferating cells p53 stabilization p21-CDK, Cell cycle arrest Apoptosis Cancer cell death Normal cell resistant Proteasome as a drug target Velcade: Bortezomib The first drug targeting proteasome Anti-cancer (multiple myeloma and some lymphoma) Cancer cell survival requires proteasomal degradation of proteins Partially inhibits proteasome, affecting a broad range of signaling Pathways Cross-talk and integration in signaling P53-MDM2 inhibitor nutlin-2 Synthesis of E3 inhibitor Nutrin-2 binds to p53 pocket, inhibiting MDM2 binding. How about targeting other ~500 E3s? Herceptin (Trastuzumab) Monoclonal antibody to HER2 Women with breast cancer Epidermal growth factor receptor-2 (HER2) Constitutively active mutant by dimerization Ras MAPK c-Jun Cyclin CDK Rb E2F G1S progression Each student is required to present a slide shown below at the end of class Please use propagated materials, websites, or slides themselves The Cell Cycle M-phase: mitosis and cytokenesis Interphase G1 (Gap1) S: DNA replication G2 G0: quiescent or senescent Homologous chromosomes Sister chromatids (replicated) G1 S G2 M kinetochore Cell cycle: G1-S-G2-M Microtubule kinesin G1 G2 Mitosis Prophase Anaphase mm pp mp + mp Metaphase Telophase Mitosis G1 The cell cycle controlled by inhibition for DNA integrity Cyclin-dependent kinase (CDK): key molecule cyclin: activator Kinase Phosphatase Ubiquitin-dependent proteolysis Core cell cycle machinery: CDK and Cyclin Cyclin-dependent kinase (CDK): key molecule Cyclin: activator Wee1: inhibitory kinase CAK: CDK-activating kinase CDC25: phosphatase APC-dependent ubiquitylation of cyclin Mitosis (M-phase) controlled by CDK and cyclin The ubiquitin system Phosphorylation vs. Ubiquitylation The 26S Proteasome Universal proteolytic machinery 19S + 20S + 19S = 26S Valcade: proteasome inhibitors as anti-cancer drug Receptor tyrosine kinase Growth factors: EGF, PDGF etc RTK signaling and Ras GTPase MAPK cascade in RTS-Ras signaling RTK: kinase receptor Ras: GTPase MAPKKK: kinase MAPKK: kinase MAPK: kinase Transcription factors (e.g., c-Fos) Why multi-step in signaling? Signal amplified in number and duration Signal fine regulated Signal cross talks Rb in G1/S progression Rb Retinoblastoma sensitivity protein CDK substrate E2F transcription factor Rb-E2F: inactive Rb-P + E2F G1/S protein expression Cell cycle checkpoint (G1/S) Cell cycle arrest when DNA damage found p53: transcription factor Cell cycle check point protein p53 phosphorylation p21: CDK inhibitor DNA repair Apoptosis G1/S checkpoint by p53 How is p53 activity induced by DNA damage? DNA damage and cell cycle arrest DNA damage DNA kinase p53 phosphorylation p21: CDK inhibitor DNA repair Apoptosis p53 mutation in >50% of cancers Mitochondrial apoptotic pathway Mitochondria: aspiration Pore: cytochrome c release Pore forming: Bax, Bak Pore facilitator: Bad, Bid Anti-pore: Bcl-2 Apoptosome Cytochrome c Apaf-1 Pro-caspase 9 Caspase 9 Apoptosis p53: cell cycle arrest and apoptosis The transcription factor p53 The ubiquitin ligase MDM2 p53 degradation DNA damage ATM, ATR kinases p53 phosphorylation and stabilization Cell cycle arrest (p21) and apoptosis Carcinogenesis Cancer and angiogenesis VEGF (vascular endothelial cell growth factor) FGF (fibroblast growth factor) Blood vessel induction into tumor mass Nutrition and oxygen supply Metastasis uPA (Urokinase plasminogen activator) Plasminogen - plasmin Procollagenases Collagenases Extracellular matrix breakdown Secreted proteases and metastasis HPV targets p53 and pRb to induce cell proliferation and to inhibit apoptosis E6 p53 (degradation) E7 Rb (binding) Apoptosis Checkpoint G1/S