Cellular Oncogenes, Growth Factors, and Cytoplasmic Signalling Circuitry Programs

Hallmarks of Cancer

  • Resisting cell death
  • Inducing angiogenesis
  • Sustaining proliferative signaling
  • Enabling replicative immortality
  • Evading growth suppressors
  • Activating invasion and metastasis
  • Reference: Hanahan, D. and Weinberg, R.A. (2000) The hallmarks of cancer. Cell, 100, 57-70.

Principal Questions on Oncoproteins

  • How can a single oncoprotein, coded by an oncogene, alter so many different cellular regulatory pathways?
  • The answer lies in how normal cells regulate their growth and division.
  • Normal cells receive growth-stimulatory signals, process them via complex circuits, and decide whether growth and division are appropriate.
  • These signalling processes relate to cell-to-cell communication, which arose with multicellular organisms.
  • Communication depends on cells emitting and receiving signals, and responding specifically.

Growth Factors (GFs) and Cell Communication

  • Growth factors are small proteins released by some cells that carry biological messages to others.
  • Decisions about growth vs. no-growth are made for the welfare of the entire tissue and organism.
  • Growth factors tie cells within a tissue together, enabling continuous communication.
  • Neighbours may provide growth-stimulating (mitogens) or growth-inhibitory factors.
  • Oncoproteins can take charge of the cell’s natural growth-stimulating machinery, mimicking the encounter of growth factors.
  • Factors secreted by nearby cells determine the behavior of neighboring cells.

Blood Clotting, Wound Healing, and PDGF

  • Platelet-derived growth factor (PDGF) is stored in secretory exocytotic vesicles called α-granules within platelets.
  • When platelets activate during clot formation, these vesicles release mitogens and survival factors.
  • PDGF are peptides that promote movement and proliferation.
  • Blood platelets adhere to form a matrix, contracting and trapping cellular components. The remaining serum contains growth factors that stimulate cell multiplication.
  • PDGF attracts fibroblasts to the wound site.
  • Cancer likened to a wound healing process that cannot stop.

PDGF and Fibroblasts

  • PDGF is a potent attractant and mitogen for fibroblasts.
  • Experiments demonstrate wound healing action with PDGF, showing proliferation in the gap of a scratched monolayer. Mutated PDGF receptors prevent gap filling.

ErbB Signalling Network

  • Illustrates how cells communicate with their surroundings using various ligands (LPA, TGF-α, EGF, etc.) and receptors (HER1, HER2, HER3, HER4).
  • The network involves input layers (receptors), adaptors and enzymes, signalling cascades (hidden layers), and an output layer (transcription factors).
  • This leads to adhesion, differentiation, apoptosis, and migration.

Signalling Cascades

  • Cells use circuits of interconnected proteins to pass signals from upstream sources to downstream targets, avoiding inadvertent activation of other signalling proteins.

Specificity and Speed of Signalling

  • How signalling pathways can exchange signals specifically with intended partners and rapidly.
  • Oncoproteins often create cancer by generating signalling imbalances.
  • Cancer is a disease of aberrant signal processing, not just inappropriate cell proliferation.
  • Individuals at the top of a hierarchical organization exert more influence.
  • Proteins already present in the cell rapidly convey mitogenic signals from the receptor to transcription factors.
  • Changes in protein structure, configuration, and intracellular localization play a dominant role.

Activation of Immediate Early Genes

  • Activation is rapid (~30 min) and does not require transcription.
  • Process involves growth factors binding to receptors, leading to alterations in the cytoskeleton, activation of translation factors, and transcription in the nucleus.

Src and Tyrosine Kinases

  • Src is a tyrosine kinase. The phosphorylation state of many proteins is altered in cells transformed by the Rous sarcoma virus.
  • Signalling through tyrosine phosphorylation is largely used by mitogenic signalling pathways in mammalian cells.
  • Protein phosphorylation on serine, tyrosine, and threonine residues – are important in making cells cancerous.

EGF Receptor

  • EGF binds to cells whose growth it stimulates. The EGF receptor was purified and sequenced from epidermoid carcinoma where it is overexpressed.
  • The EGF receptor functions as a tyrosine kinase. Its ectodomain binds EGF and activates the cytoplasmic domain, which has sequence similarity to the Src oncogene.
  • Src oncogenic virus is contained in EGF, thus making this receptor oncogenic.

Receptor Tyrosine Kinase (RTK) Families

  • Lists various RTK families including Ros, ErbB, Ins, PDGF, VEGF, FGF, PTK7, Trk, Ror, MUSK, Met, Axl, Tie, Eph, Ret, Ryk, DDR, LMR, and ALK.
  • Highlights the different domains (Tyrosine kinase, Cysteine-rich, Fibronectin type III, Leucine-rich, Cadherin, Discoidin, Ig, EGF, Kringle, SAM, Psi, WIF, Ephrin binding domain, Fz, Ldla propeller, YWTD, Acid box, Sema, and Mam) within these receptors.
  • There are 58 in human genome.

Receptor Dimerisation and Cross-Phosphorylation

  • RTKs bind growth factors, dimerize, and cross-phosphorylate each other.

erbB Oncogene

  • erbB discovered in avian erythroblastosis virus (AEV), induces leukemia of red blood cell precursors.
  • erbB is homologous to EGF receptor but lacks ligand binding ectodomain sequences.
  • The truncated EGF receptor sends signals constitutively.

Deregulation of Receptor Firing

  • Altered structure or expression levels; regulation of receptor turnover on the plasma membrane through endocytosis.

Gene Fusion and Constitutively Dimerised Receptors

  • Gene fusions result in truncation of the ectodomain and fusion with proteins prone to dimerisation or oligomerisation.

Constitutive Active Mutant Forms of Kit

  • Mutations alleviate the suppression of receptor firing in stem cell factor (SCF) signaling.

Auto-stimulatory or Autocrine Signalling

  • Normally, cells do not synthesize a growth factor ligand for their receptor.
  • In invasive human breast carcinoma, cancer cells are surrounded by non-staining stroma; EGF receptor (red) and TGF-α (green).

Paracrine, Endocrine, and Autocrine Signalling

  • Paracrine signalling: signals sent from one cell type to nearby cell type.
  • Endocrine signalling: signals sent through circulation from cells to a distant tissue.
  • Autocrine signalling: auto-stimulatory loop where cells produce their own mitogens.
  • Normal tissues require signals from neighbors, ensuring stability and tissue architecture. Self-reinforcing positive feedback loops can lead to physiological imbalances.

Domain Structure of Src

  • SH1: tyrosine kinase catalytic core
  • SH2: binding domain for specific oligopeptide sequences flanking a p-tyr on its C-terminal site. (117 in human genome)
  • SH3: binds proline-rich sequence domains in partner proteins

Structure and Function of SH2 Groups

  • SH2 domain works as a modular plug, with binding sites for phosphotyrosine and flanking amino acids.

Attraction of Signal-Transducing Proteins by Phosphorylated Receptors

  • EGF and PDGF receptors attract various signal-transducing proteins upon phosphorylation.

SH2 and SH3 Domains

  • Linked to proteins like Fps, Src, Syk, GAP, PLC-y, and bridging proteins (adaptors) such as Grb2 and Nck.

Molecular Ligands and Their Binding Domains

  • Modified peptides (p-Tyr, p-Thr, p-Ser, Me-Lys, Ac-Lys, Ub, Ubn) bound to SH2, PTB, FHA, 14-3-3, WD40x8, MH2, Chr, Bromo, UIM, and UBA domains.
  • Peptides (NPXY, RXXK, PXXP, PPXY, FPPPP, Pro D/E-XXLL Val-COOH) interact with PTB, SH3, EVH1, GYF, VHS, PDZ, PUM, Tubby, and WW domains.
  • Domain/domain interactions via PDZ, SAM, DD, DED, CARD, PyD, PB1, and BRCT domains.
  • Phospholipids (PI(3,4,5)P3, DAG, PI(4,5)P2, PA/PS, PI(3)P) bound by C1, PH, FYVE, FERM, C2, Tubby, PX, and ENTH domains.

Ras Signalling Pathway Discovery

  • Discovered in the Drosophila eye using sevenless (homolog of EGF receptor).
  • Involves RTK, adaptors (Shc, Grb), Sos (GEF), and Ras.

The Ras Signalling Cycle

  • GTP hydrolysis and Ras inactivation induced by GAP.
  • Upstream stimulatory signals and Ras activation triggered by GEF.
  • Oncogenic Ras mutations cause blockage.

Association of Sos with Growth Factor Receptors

  • Accomplished through adaptors like Grb2 and Shc.

Downstream of Ras, the MAPK Pathway

  • Leads to tumourigenicity, cell proliferation, anchorage-independent growth, loss of contact inhibition, and cell shape changes.
  • Involves HMG14, H3, Fos/Jun (AP1), HB-EGF, CycD1, Fos, and P21Waf1.

The AKT/PKB Pathway

  • Controls cell proliferation, cell death and cell growth.
  • Involves PI3K, PIP3, Akt/PKB, Rho-GEFs, Ras, Ral-GEF, Raf (MAPKKK), MEK (MAPKK), Erk1/2 (MAPK), mTOR, and Bad.
  • Inhibition of apoptosis, stimulation of protein synthesis, proliferation, with the influence of Cdc42, and Rac related to filopodia and lamellipodia.

Ral and the Control of Cytoskeleton

  • Rac emits mitogenic signals and antagonizes Rho proteins through ROS.

Downstream of Ras, the Ras Effector Loop

  • Loop’s function is impacted by GTP and various residues (Y40, G12V, T35, Y40C, E37, T35S, E37G) influencing PI3K, Raf, and Ral-GEF.

Minor Signalling Lipids with Inositol Headgroups

  • Lipids in the bilayer with hydrophilic inositol headgroups and hydrophobic lipid tails.

Enzymatic Modification of Phosphatidylinositol (PI)

  • Involves PI3K, phospholipase C (PLC), and kinases modifying PI to phosphatidylinositol-(4,5)-diphosphate (PIP2) and phosphatidylinositol-(3,4,5)-triphosphate (PIP3).

Docking of PH Domain on Akt/PKB to PIP3

  • Leads to Akt/PKB activation involving P13K, PIP3, PTEN, PDK1, and PDK2.

Akt/PKB Phosphorylates Downstream Targets

  • Phosphorylates GSK-3β, HIF-1a, and Bad, influencing proliferation, angiogenesis, and apoptosis.

Conceptual Representation of a Signalling Network

  • Input layer (RTKs) influences core processes (PI-3K, MAPK, Ca2+ signaling).
  • Feedback processes define emergent properties.
  • Output layer (transcriptional responses, cytoskeletal changes) is read out by core processes.

Modulation of Signalling Molecules

  • Intrinsic activity:
    • Noncovalent modification (e.g. binding of GTP, phospholipid, Ca2+Ca^{2+})
    • Receptor dimerisation
    • Covalent modification (e.g. phosphorylation)
    • Proteolytic cleavage
  • Concentration of a signalling molecule:
    • Transcriptional
    • Protein stability
  • Intracellular localisation
  • Kinetics
  • Activation of dormant signalling molecules
  • Modular architecture
  • Afferent (incoming) signals
  • Efferent (outgoing) signals, responses

Thought Questions

  • Why is autocrine signalling an intrinsically destabilising force for a normal tissue?
  • How might a cell be altered to respond to one growth factor (e.g. EGF) with responses characteristic of another (e.g. PDGF)?
  • What mechanisms might cells use to reduce responsiveness to growth factors over time?
  • What evidence supports the notion that growth factor receptor firing depends on receptor dimerisation?