1 Intracellular Signaling and Control of the Cell Cycle

2 Intracellular signalingPathways initiated by ligand binding to receptors, usually plasma membrane proteins Modulate cell growth, metabolism, death, differentiation, movement, and invasion First link between intracellular signaling and cancer development came from studying RNA and DNA viruses in animals Ex: src, ras, myc, sis, akt, raf RNA retroviruses Other ex: DNA tumor viruses that inactivate tumor suppressor genes like Rb and p53

3 Implications of delineating signal pathways: defining and targeting pathways can result in targeted therapies that are more potent and less toxic

4 Intracellular signalingTaking cues in the extracellular environment and relaying/interpreting them in a cell External cues: growth factors signaling division of a cell ECM proteins to promote survival cytokines to induce differentiation Other signals to promote motility and invasion

5 Intracellular signaling: complexOne receptor can engage many different pathways One pathway can influence another (cross-talk) Same ligand can have different effects in different cell types Activating a receptor usually also turns on negative regulatory mechanisms

6 Steps Receptor activation by ligandDimerization/phosphorylation activates an inactive cytoplasmic portion Initiation of signal cascade (by adaptor/scaffolding proteins) Up/down regulation of gene transcription or expression Induction of negative regulators to switch off pathway

7

8 Receptors Most found in plasma membrane, work for peptide and protein ligands that cannot cross lipid bilayer Some receptors are in the cytoplasm and nucleus, for ligands that are lipid soluble and can cross membrane (ie steroid hormones)

9 G-protein coupled receptorsLargest class of plasma membrane receptor All share a common architecture, 7 transmembrane a-helical domains, connected by intra- and extra-cellular loops Ligand binds the extracellular domain  induces conformational change  G protein binding the intracellular domain becomes activated Activation causes dissociation of the a subunit from the beta-gamma domain, both parts go on to mediate other signaling events

10 Receptor tyrosine kinasesMost involved in control of cell growth, motility, and differentiation and metabolic control Examples: PDGF-R, EGFR, hepatocyte growth factor receptors, FGF-R, insulin receptors Some have known ligands, some don’t have ligands (ex: Her2 aka ErbB2) – signals by heterodimerizing with other receptors Majority are single polypeptide chains with EC ligand-binding domains, short transmembrane domains, and cytoplasmic region with the kinase domain

11 RTKs General mechanism: ligand binds, receptor dimerizes/oligomerizes, two catalytic domains are brought together, each domain phosphorylates tyrosine residues in the activating loop of the other, conformational changes arise to allow binding sites for enzymes and adaptor proteins to induce further signaling

12 Serine/threonine kinase receptorsAka TGF-B superfamily of receptors Ligands: TGF-B, activins and inhibins, and bone morphogenetic protein Involved in development and morphogenesis as well as cell cycle progression, wound healing, motility, immune surveillance Ligands interact with two single-pass membrane proteins (type I and II receptors) Cytoplasmic domains have intrinsic serine/threonine kinase activity (phosphorylate serine and threonine residues)

13 Serine/threonine kinase receptorsLigand binds  2 type I receptors oligomerize with 2 type II receptors  Type II receptor phosphorylates and activates type I receptor  Phosphorylates substrate proteins like Smad

14 Integrin receptors Heterodimers of alpha and beta subunit, each is membrane spanning Can bind ligands outside the cell as well as cytoskeletal components inside the cell, integrating the two Without ligands, EC beta subunits are “bent” Ligand binds  beta subunit straightens  conformational change of transmembrane and cytosplasmic domains  signal via cytoplasmic kinases (like Src and FAK), do not have intrinsic kinase activity

15 Integrins Unique: can do “inside-out” signalingCytoplasmic pathways (ie by activation of EGFR or Frizzle pathways) cause conformational change in EC domain  increased adhesion to EC ligands and subsequent outside-in signaling Requires the cytoskeletal protein talin

16 Cytokine receptors Superfamily consisting of receptors for growth hormone, erythropoietin, thrombopoietin, G-CSF, interleukins, interferon Ligand binding  dimerization  receptors have no catalytic activity JAK kinases are associated with the cytoplasmic domains  transphosphorylate receptors  initiates signaling via Stat phosphorylation

17 Cytokine receptors One type is TNF superfamily of cytokine receptorsAKA death receptors, control apoptosis in response to exogenous signals Ex: TNF receptors 1 and 2, Fas receptors Have EC ligand-binding sites, transmembrane protein, and death domain on intracellular side Ligand binding can cause cell to undergo apoptosis

18 Others Frizzled receptors are binding partners for Wnt ligands, control development processes/stem cell fates and are implicated in cancers Notch receptors important for development and tissue homeostasis. Specify cell fates and create boundaries between cells. Ligands are DSLs which are also transmembrane proteins. Signaling occurs when one cell with Notch receptor comes close to another cell with DSL ligand. Intracellular domain of Notch receptor can go to nucleus directly and activate transcription (does not use signal transduction pathways). Nuclear hormone receptors also do not not require signal transduction pathways, ligand can go directly to nucleus. Ligands include estradiol, progesterone, androgens.

19 Components: Adaptor proteinsProteins used in signaling cascade Can act as scaffolds to group together signaling molecules Can give specificity to signaling by clustering sets of signaling proteins in a certain cellular location Some adaptor proteins are selective for certain receptor-signaling systems, ex: death domain-containing adaptors for TNF superfamily of receptors

20 Intracellular ComponentsCytoplasmic tyrosine kinases For receptors that don’t have intrinsic TK activity Subfamilies of cytoplasmic TKs: Src family, Abl family, FAK family, JAK family Cytoplasmic serine and threonine kinases Phosphorylate serine/threonine residues Ex: MAP kinase pathway MAP kinases are single subunit serine/threonine kinases that translocate to the nucleus when activated Best known example: Ras-Raf-MAPK pathway Ras is a GTPase, usually inactive in unstimulated cells

21 Raf: a serine/threonine kinase

22 mTOR: activated by S/T kinasesAkt, ERKs, and other S/T kinases inactivate the tumor suppressor complex TSC1/2 This allows activation of mTOR  signaling allows mRNA translation

23 Negative regulators After ligand binding, receptors internalized via clathrin-coated pits and endocytosis  degradation in lysosomes Receptor can also be recycled back to the cell membrane Can undergo ubiquitin-mediated degradation Self inhibition: activation of MAPK pathway activates enzymes that dephosphorylate and inactivate MAPK Phosphoprotein and phospholipid phosphatases can regulate signaling Ex: PTEN (tumor suppressor) is a phosphoinositide phosphatase; loss of this can result in malignancies such as GBM and prostate cancer

24 Control of the Cell Cycle

25 Most cells in the body are in G0 state (quiescent state)Most are terminally differentiated and don’t divide Certain populations retain ability to divide throughout life: hematopoietic cells, cells of the gut – have high proliferation rates

26 To proliferate or not? Tightly regulated Exogenous signalsPro-proliferation: nutrients, mitogenic growth factors (EGF, PDGF) Negative: inhibitory growth factors (TGF-B) Also influenced by interaction with the ECM Disruption of balance can reduce/expand cell populations Tumors have mutations that impair pathways that suppress proliferation or activate pathways that promote proliferation

27 CDKs and checkpoints Proliferating cells must copy genes and distribute to daughter cells with high fidelity 2 mechanisms ensure proper division CDKs (cyclin dependent kinases): evolutionarily conserved enzymes that control cell cycle transitions Cell cycle checkpoints: surveillance pathways that monitor for errors/DNA damage and activate cell cycle arrest and DNA repair mechanisms Most tumors have mutations in both CDK and checkpoint pathways Can be useful by exploiting sensitivity to chemotherapeutic agents that induce DNA damage

28 Cell Cycle Overview

29 Cell cycle phases Go – quiescent G1 – “cell growth” phase (8-30 hrs)Upregulation of transcription of proteins that regulate division. Doubling of macromolecules for partitioning to each daughter cell. Requires mitogenic growth factors. Loss of these will stop division. Restriction point – committed to divide, now growth factor independent. S phase – synthesis phase Chromosomes are each replicated once G2 – second gap phase (3-5 hrs) M phase – mitosis

30 CDKs Large subfamily of conserved serine/threonine kinases that regulate the cell cycle Defined by their dependence on cyclins

31 Cyclin-CDK complex

32 Expression of cyclins and CDKs is restricted to specific stages of the cell cycleG1: regulated by CDK4 and CDK6 G1 to S transition and S phase: CDK2 G2 and mitosis: CDK1

33

34 Cyclin classes: D, E, A, B typesClassified based on cell cycle stage on which they act

35 D-type cyclins Promote cell cycle entry in 2 ways:Present at low levels in quiescent cells Expression induced during G1 by mitogens and then persists through all cell cycle stages Mitogens induce different D-type cyclins Signaling via EGF/ras and Wnt/B-catenin induces cyclin D1 C-myc induces cyclin D2 Each D cyclin is probably functionally redundant, more important is total level of D-cyclins in the cell Promote cell cycle entry in 2 ways: D type cyclins move CDK inhibitors away from other CDK complexes, allowing their activation D1, D2, D3 associate with CDK4 and 6 and phosphorylate pRB (retinoblastoma protein), a gatekeeper for cell cycle re-entry

36 E-type cyclins Expressed late in G1 under control of E2 transcription factors Cyclin E binds specifically to CDK2 Complex is required for cells to move through G1 to S phase transition Cyclin E/CDK2 phosphorylates substrates to help cell progress Increases histone pools to package replicated DNA Induces duplication of centrosomes that form the mitotic spindle Can also phosphorylate subtrates to inhibit cell cycle Can phosphorylate pRB and inhibit its suppressive function Can activate SCF (a ubiquitin ligase) which ubiquitinates cyclinE/CDK2 and causes its degradation – this ensures the action of cyclin E/CDK2 is restricted to one phase of the cell cycle

37 A-type cyclins Transcribed during late G1 under control of E2F transcription factors Associates with both CDK2 and CDK1 Required for S phase progression Enters the nucleus during S phase and localizes at nuclear replication foci Acts during G2 and at beginning of mitosis Initiates condensation of chromatin and may activate cyclin B/CDK1 complexes Destroyed during mitosis via a ubiquitin ligase called anaphase promoting complex (APC)

38 B-type cyclins Appear at beginning of G2, accumulate steadily throughout G2 Associates with CDK1 Cyclin B1/CDK1 kept inactive throughout G2 by Wee1 kinases and inhibitory phosphorylation Activation occurs during prophase in mitosis Activation mediated partly by CyclinB1/CDK1 itself: small amount of activated molecule inactivates its own inhibitors and activates its activators  feed forward loop Complex initiates centrosome separation to form the mitotic spindle, then translocates into nucleus to orchestrate mitosis Degraded at the end of metaphase by APC Degradation of CDKs is required for cytokinesis (separation of daughter cells) and re-entry into Go/G1

39 CDK Inhibitors Can prevent cell division in response to external signals or internal stresses Activity of CDKIs is promoted by growth suppressive signals (ie TGF-B) and inhibited by proliferative signals 2 families: INK4 and CIP/KIP INK4 targets CDK4 and CDK6 Alterations in INK4 seen in many human tumors Ink4a is a tumor suppressor. Can be inactivated by point mutation/deletion/methylation in 30% of all human tumors, also associated with familial melanoma

40 CDK inhibitors CIP/KIP family inhibits activity of cyclinE/CDK2 and cyclinA/CDK2 kinases Can also paradoxically promote cyclinD/CDK4/6 complexes Balance of these two activities determines if cell will divide or not under stress

41 Increase in mitogens boosts cyclin D levels, which exceed INK4 levels Overall, cell cycle progression is determined by balance/tipping point between CDKIs D type cyclins G0/G1 cells have high levels of CDKIs and low levels Cyclin D  cell cycle entry is blocked Increase in mitogens boosts cyclin D levels, which exceed INK4 levels Cyclin E/CDK2 complexes then accumulate and pRB is phosphorylated and inactivated  TIPPING POINT in commitment to cell cycle entry

42 pRB Classic tumor suppressorInhibits entry into cell cycle by inhibiting E2F transcription factors To enter cell cycle  must phosphorylate pRB and cause it to dissociate from E2F Inactivation of pRB is essential step in tumorigenesis Tumors with wild type pRB often have mutations downstream in cyclin D1 or CDK4

43

44

45 Cell cycle checkpointsSignaling pathways make sure events in one phase are complete before going to the next Occur at 4 points in the cell cycle: G1/S Intra-S G2/M Metaphase to anaphase transition (aka spindle checkpoint)

46 DNA damage response (DDR)Checkpoints monitor structure of chromosomal DNA during cell cycle progression Scan chromatin for partially replicated DNA or strand breaks Complexes recruited to repair DNA and activate signaling pathways to induce temporary cell cycle arrest

47 DDR Two components Two members of PI3K related family: ATM ATREarlier thought that ATM responded to double stranded breaks and ATR responded to single stranded breaks Really more complicated with cross talk between the two Mutations of ATM found in hereditary ataxia-telangiectasia (cancer predisposition and radiation hypersensitivity) DDR affected in many hereditary syndromes and human tumors (BRCA1/2 and Fanconi’s anemia) ATM/ATR also activate p53 to transcribe pro-apoptotic genes

48

49

50 Spindle checkpoint

51

52 Cell cycle deregulation in cancerMost frequent alterations in cell cycle machinery: loss or mutation of pRB tumor suppressor Overexpression of cyclins/CDKs Loss of expression of CDKIs Most frequently altered cell cycle checkpoint signaling molecule: p53 tumor suppressor Proteins upstream of p53 (ATM and CHK2)

53

54 Therapeutic manipulation?Can you target CDKs? Activity elevated in many tumors but also essential to maintain normal cell populations in adults (hematopoietic cells and gut) Some promising results in mouse models but cell cycle machinery also responds robustly by substituting other CDKs for the missing one tumors may develop rapid resistance to these Efforts focus on creating specific CDK inhibitors or pan CDK inhibitors

55 Flavopiridol Pan CDK inhibitor Arrests cells at G1/S and G2/MFavorable responses in phase I and II studies in renal, colorectal, gastric, lung, esophageal cancers Can also use chemo (Gemzar) to target cells in S phase and then treat with flavopiridol  enhanced cytotoxic effect Can also use Taxol to inhibit mitotic spindle function and then treat with flavopiridol for synergy

56 Targeting DDR systems PARP inhibitors – selectively kill cells lacking BRCA1 OR 2 BRCA1 /2 and PARP all provide alternative repair mechanisms for DNA damage Loss of one but not both repair mechanisms can be tolerated