Oncogenes

• What is cancer?

  The accumulation of mutations in a stepwise process
• What is an oncogene?

  • A gene which encodes a protein able to transform cells
  • Develop from normal self-genes (known as proto-oncogenes) that normally function to control cell growth
• How are proto-oncogenes converted to oncogenes?

  Gain of function mutation

  1. Point mutation (mutated DNA affects encoded protein) - increased activity
  2. Gene amplification (when DNA polymerase copies the DNA template which slips) - more protein
  3. Chromosomal translocation (when a chromosome section breaks off and switches with another) - breaks due to chromosomal translocation can disrupt genes, also different chromosomal locations experience different rates of transcription
• What are 3 main examples of proto-oncogenes?

  1. Src (proto-oncogene c-Src)
  2. EGFR (epidermal growth factor receptor)
  3. Ras (rat sarcoma virus)
• What is Src?

  • Non-receptor tyrosine kinase
  • Intracellular signalling molecule that drives cell growth
• What are the domains of c-Src?

  • SH3
  • SH2
  • Catalytic kinase domain
  • C-terminal Tyr (Y) residue
• What is the structure of c-Src in resting cells?

  • c-Src is phosphorylated at C-terminal Tyr (becomes pTyr) - unique exception as phosphorylation normally activates cells
  • Causes protein to fold - Tyr interacts with SH2 domain (while SH3 domain binds Pro residues)
  • Folded strcuture = masks the active site of kinase domain so it cannot phosphorylate anything

  
• What is the structure of c-Src in activated cells?

  • Activated phosphatase cleaves phosphate from terminal Tyr
  • Protein unfolds
  • Various domains can now interact with residues in other substrates - interacts with Tyr residues in substrates and phosphorylates them to pass on growth signal (occurs in presence of GFs and activated receptors)

  
• What made v-Src oncogenic and c-Src not?

  Phosphorylation site is removed (C-terminal Tyr cleaved off)

  Without it, protein cannot fold (and be in its inactive form)

  Thus protein is remains open (constitutively active)
• What happens when cells are transfected with mutated c-Src?

  Cells are a uniform size/shape --> Cells round up

  Monolayer, neat arrangement --> Form a mass and pile up

  Default growth speed --> Faster growth
• What allows oncogenic Src to continue to pile up and grow?

  There is no contact inhibition

  • Rounded phenotype = much less cell-cell contact than normal phenotype
  • Easier for cells to break away = key in metastasis where cells break away from the primary tumour and recolonise new areas
  • Less tight junctions holding cells together
• How do you test for oncogenic function?

  Check the ability of transfected mutated DNA to transform cells and change characteristics towards malignancy
• What mechanism do inhibitors use to target oncogenic Src?

  • As a tyrosine kinase, Src is reliant upon ATP as a source of phosphate (to phosphorylate its substrates)
  • Small molecule inhibitors block the ATP on the active site and starve Src of phosphate
  • Src cannot phosphorylate anymore
• What inhibitors have been developed against Src?

  1. Dasatinib
  2. Saracatinib
  3. Bosutinib
• What are the domains of EGFR?

  • Extracellular ligand-binding domain - upon ligand binding, conformational changes occur in the extracellular domain that promote receptor dimerisation
  • Transmembrane domain
  • Intracellular (cytoplasmic) tyrosine kinase domain - activated upon ligand binding, leading to autophosphorylation of specific tyrosine residues within receptor itself and phosphorylation (activation) of downstream signalling pathways, e.g. Ras/MAPK for cell proliferation, PI3K-PKB for survival (anti-apoptotic)
• How is EGFR mutated?

  Same receptor but 2 different modes of mutation

  
  

  Point mutations

  • Makes receptors ligand independent (active without ligand)
  • Chains come together and interact in absence of ligand and signal continuously
  • Point mutation = activation of the receptor itself

  Gene amplification

  • Receptors still ligand dependent (but with more receptors)
  • Template slippage = more gene copies = more protein copies = more receptors (overexpression)
  • Cells become sensitive to low levels of GF
  • Overexpression = activation of downstream pathways (rather than receptor itself)
• Describe EGFR signalling in cancers (Val-->Gln mutation)

  Ligand independent - leads to lung cancer

  1. Val residue (hydrophobic) between transmembrane region can be mutated to Gln (+ve charged) - no longer sits well inside lipid membrane
  2. The 2 chains of the extracellular ligand binding region now dimerise to hide the positive charge of Gln
  3. This brings together the intracellular singalling kinase domains which activate each other and induce signalling pathways

  
• Describe EGFR signalling in cancers (deletion mutation)

  Ligand independent - leads to lung cancer

  1. Extracellular ligand binding region is cleaved off - remaining domains become unstable
  2. Transmembrane domain and intracellular signalling kinase domain dimerise in the absence of ligand to activate each other and induce signalling pathways

  
• Explain the agents used against breast cancer

  Ligand dependent (not constitutively active)

  • As receptor is still WT but overexpressed - use humanised monoclonal antibodies rather than SMIs

  1st generation

  • Herceptin (trastuzumab) - specifically blocks ligand binding to HER2/neu receptor (overexpressed in 25-30% breast cancers)
  • Eventually develop resistance

  2nd generation

  • Cetuximab - binds and inhibits EGFR mediated signalling pathways
  • Pertuzumab - targets the HER2 receptor via. a different epitope than Herceptin, leading to complementary inhibition of HER2-mediated signaling pathways
• Explain the agents used against lung cancer

  Ligand independent receptors (constitutively active)

  • Small molecule inhibitors

  1st generation

  • Competitive inhibitors - block ATP binding to EGFR tyrosine kinase domain
  • Block downstream signalling pathways
  • Initially effective but may develop secondary mutations in EGFR gene (e.g. T790M mutation) - inhibitor cannot bind but ATP can

  2nd generation

  • Irreversibly-bound inhibitors - bind multiple ErbB family receptors
  • Broader spectrum

  3rd generation

  • Irreversible covalent binding to EGFRs specific to T790M mutation - prevent binding of ATP
• Why are patients with declined cardiac function not suited to antibody treatment?

  Blocking Her2 receptors in the heart can cause cardiotoxicity and worsen the declined cardiac function
• Summarise the mechanism of actions of small molecule inhibitors as cancer agents

  Small molecule inhibitors

  • Block ATP binding site
  • Kinase has no source of phosphate for phosphorylation
• Summarise the mechanism of action of antibodies as cancer agents

  Largely antibody-dependent cellular cytotoxicity (ADCC)

  • FcR receptors on NK cells recognise Fc region of antibodies
  • Leads to apoptosis of Her2+ cells

  May also induce downregulation of receptor (less Her2) and thus downstream signalling

  • Switches off Src activation (drives cell growth) = decreased growth
  • Increases inactivation PI3K/PKB pathways (promotes cell survival) - induces cell cycle arrest, increased p27 (CDKI)
  • May increase apoptosis, decrease angiogenesis
• What is the Ras-MAPK pathway?

  • Drives cell growth
  • Downstream of EGFR
• What is Ras?

  Ras is a GTPase (hydrolyses GTP to GDP) and activates Raf further down the pathway
• What are common mutations of Ras?

  Point mutations occur within certain hotspots within the sequence

  • Activating mutations at position 12 and 61
  • Mutations can differ between cancers (e.g. aa12 VtoG in bladder but VtoS in lung cancer)
  • Need different therapetuics for different Ras mutations
• Why is Ras considered an oncogene?

  • Loss of function mutations generate constitutively active Ras proteins
  • Constant signalling to Raf = excessive activation of growth pathway as ERK translocates to nucleus = upregulates cyclin D
• Summarise the mechanism of action of Ras anti-cancer agents

  Small peptides

  • Ras is tethered to the membrane via. PTMs (fatty acid modifications)
  • Farnesyl transferase inhibitors (peptidomimetics) block this modification - i.e. block farnesyl transferase from adding farnesyl groups
  • Ras cannot be localised to the membrane - falls off and unable to interact with Raf
• How is the cancer metastasis potential determined?

  Three different theories

  1. Clonal selection
  2. Predetermined genetic predisposition
  3. The host predisposition
• Cancer metastasis potential theory 1: Clonal Selection

  Linear model

  • Based on natural stepwise progression of cancer
  • Only advanced neoplasms have enough mutations to facilitate dissemination from primary tumour
  • Late tumourigenesis event

  Parallel model

  • Metases develop in parallel (simultaneously) to the primary tumour - but acquire independent (epi)genetic alterations
  • Early tumourigenesis event

  Classical model

  • Rare tumour cells acquire mutations that increase over time due to selective advantage - metases form from these specific clones
  • Late tumourigenesis event

  
• Cancer metastasis potential theory 2: Predetermined Genetic Predisposition

  • Metastatic potential (mutations) is (or is not) expressed in the majority of cells in primary tumours - an intrinsic property of each tumour from the start
  • Only these particular primary tumours are preconfigured to metastasise
  • If so, metastatic potential should be predictable from the 1° (primary) cancer profile
  • Metastatic potential hardwired into early tumours

  
• Cancer metastasis potential theory 3: Host Predisposition

  • The host genotype is predisposed (or not) to be metastasis permissive
  • Tumours arising in individuals with a certain host genotype have a higher probability of metastasis
  • This is genotype influence on metastatic potential is distinct from genetic susceptibility to cancer itself which is due to highly penetrant rare mutated alleles (or defective suppressor genes such as BRCA1)

  
• Rous Sarcoma Virus (RSV) and Human Papillomavirus (HPV)

  RSV

  • Retrovirus – RNA as genetic material (uses RT and IN)
  • Associated with sarcoma
  • Carries v-src oncogene derived from c-src (proto-oncogene) – v-src = constitutively active, promotes cell proliferation and differentiation
  • No vaccine

  HPV

  • DNA virus – DNA as genetic material
  • Associated with various cancer – e.g. cervical, anal
  • Expresses two major oncogenes, E6 and E7 – disrupt normal function of tumour suppressor proteins p53 and Rb respectively, promotes cell growth
  • E5 subunit causes prolonged activation of PDGFR
  • Vaccine
• What are the HPV vaccine options?

  Many different types of HPV, with something considered higher or lower risk - most contracted through sexual interactions

  • Gardasil 9 contains L1 proteins from nine HPV types (6, 11, 16, 18, 31, 33, 45, 52 and 58) - assembled into virus-like particles (VLPs) via. recombinant DNA technology
  • L1 proteins stimulate the immune system to produce antibodies against HPV - protects against HPV-related diseases without causing infection
  • HPV types 16 and 18 cause a large majority of cervical and anal cancers, while HPV types 6 and 11 cause most of genital warts
• What is epidermal growth factor receptor (EGFR)?

  • Cell surface receptors belonging to the ErbB family of receptor tyrosine kinases
  • Crucial roles in cell growth, proliferation, and differentiation
  • Dysregulation of EGFR signaling is implicated in various cancers, making EGFRs important targets for cancer therapy
• Describe general EGFR signalling

  • Ligand (e.g. EGF) binds to extracellular domain of EGFR - triggers conformational change and dimerisation of EGFR homodimer
  • Dimerisation activates the intracellular tyrosine kinase domain of EGFR - causes autophosphorylation of specific tyrosine residues within the receptor's cytoplasmic tail
  • Phosphorylated tyrosine residues on EGFR serve as docking sites for various cytoplasmic signalling molecules which initiate downstream signaling cascades - e.g. Ras/Raf/MAPK for cell proliferation and PI3K/PKB for survival (anti-apoptotic)
• How does EGFR signalling trigger Ras/Raf/MAPK pathway?

  • Phosphorylated EGFR recruits Grb2 to its intracellular cytoplasmic domain
  • Grb2 then binds SOS which acts as a guanine nucleotide exchange factor (GEF) for Ras - promotes exchange of the GDP that is bound to Ras for GTP, thus activating Ras
  • Active Ras initiates a signalling cascade by recruiting and activating (phosphorylating) Raf which further activates (phosphorylates) MEK and ERK (MAPK)
  • ERK translocates into the nucleus to drive transcription of cyclins - driving the cell cycle