Angiogenesis and metastasis

• Why do cancer treatments target angiogenesis and metastasis ?

  • Initial cancer treatments broadly targeted rapidly dividing cells but could not distinguish between normal healthy cells and cancer cells
  • Developed therapies which were more targeted, but these have more chance for resistance build up
  • Targeting cancer hallmarks like angiogenesis and metastasis will be specific to cancer, and unlikely to develop resistance (as they are key processes)
• What is angiogenesis?

  • Angiogenesis is the process of forming new blood vessels from pre-existing ones
  • Blood vessels bring oxygen and nutrients towards cells while carrying away CO2 and cellular waste
• What is the difference between angiogenesis and vasculogenesis?

  Angiogenesis: formation of new blood vessels from pre-existing ones.

  Vasculogenesis: de novo formation of blood vessels from precursor cells.

  
  

  Vasculogenesis only occurs during embryogenesis, wound healing (developed organisms), and the menstrual cycle
• How does tumour neoangiogenesis differ from angiogenesis in normal tissue?

  Tumour: Abnormal, random (not programmed), leaky

  Normal tissue: Controlled, organised, stable
• Are normal cells or tumour cells closer to a blood supply?

  Normal cells - more stable and efficient blood supply

  
  

  Disorganised nature of tumour angiogenesis means cells in the centre of a tumour mass can be pushed far away from blood vessels - centre becomes hypoxic (lacking oxygen) and eventually necrotic.

  However, cells on the outside continue to proliferate (closer to blood vessels) and contribute to tumour mass
• What kind of growth factors do tumours secrete?

  e.g. Vascular endothelial growth factor (VEGF)

  
  

  VEGF binds VEGFRs on the surface of growing endothelial cells (interior of blood vessels)

  • Activates endothelial cells and causes sprouting (of new blood vessels)
  • Decreasing concentration gradient of VEGF from the hypoxic regions of the tumour to the tumour periphery - new vessels grow towards tumour (up the gradient)

  Also e.g. platelet derived growth factor (PDGF), fibroblast growth factor (FGF), hypoxia inducible factor 1a (HIF-1a)
• How does VEGF affect endothelial cells?

  • Disorganised vascular structure
  • Low inter-endothelial cell junctions
  • Low pericyte coverage
  • Increased microvasculature permeability (leakiness)
• How does VEGF affect blood vessels?

  Vessels have low integrity --> collapse easily --> low perfusion (sufficient for tumour survival but not for administration of intravenous drugs)
• What is the difference in expression of VEGF in normal cells vs. cancer cells?

  Normal cells express minimal levels of VEGF (normally downregulated)

  
  

  Cancer cells hijack mechanism to switch on VEGF
• How is angiogenesis controlled?

  Activators (pro-angiogenic) and inhibitors (anti-angiogenic) control the angiogenic switch

  
  

  Activators = growth factors/cytokines - low levels in normal cells until detect hypoxic environments

  • VEGF
  • PDGF
  • bFGF (downregulated by p53)

  Inhibitors - in excess in normal cells to balance out activators

  • Endostatin
  • Angiostatin
  • Thrombospondin (upregulated by p53)
• How does p53 regulate angiogenesis?

  p53 is a tumour suppressor gene that controls DNA integrity but also suppresses angiogenesis

  p53 loss of function = inability to upregulate anti-angiogenic factors and raise/stabilise pro-angiogenic factors
• What happens when there are high levels of activators of the angiogenic switch?

  Tumours are likely to grow faster and have an increased risk of metastasis

  Enhanced angiogenesis = more blood vessels = more escape routes for tumour cells to enter = worse prognosis
• Which cancers are dependent on VEGF?

  Highly dependent: colorectal, renal

  Less dependent: breast, pancreatic, prostate, melanoma
• What other signalling pathways integrate with downstream signalling from VEGFRs?

  • MAPK - cell proliferation, e.g. influence endothelial cells to sprout
  • PLC/PKC - endothelial cell permeability (leaky vessels)
  • FAK - cell migration
  • PI3K - promotes cell survival, e.g. phosphorylating and inhibiting pro-apoptotic proteins (such as BAD)
• How do monoclonal antibodies target angiogenesis?

  VEGF receptor are ligand-dependent (unlike EGFR)

  
  

  Target the the ligand

  • Ligand is overexpressed by tumour cells - mop it up so it cannot bind to receptor
  • e.g. Bevacizumab (Avastin) - anti-VEGFA antibody

  Target the receptor

  • Bind to the extracellular domain of VEGFR, preventing VEGF from binding to its receptor
  • e.g. Ramucirumab - anti-VEGFR-2 antibody
  • Some level of specificity
• How do decoy receptors target angiogenesis?

  Endogenously control amount of (overexpressed) free GFs and cytokine ligands - mop up excess ligand so it cannot bind to true receptor

  • e.g. Aflibercept (Zaltrap) = recombinant fusion molecule that binds VEGF-A, VEGF-B
  • Slightly longer half-life than monoclonal antibodies (easier dosage)
• How do receptor tyrosine kinase inhibitors target angiogenesis?

  Small molecule inhibitors that bind the intracellular tyrosine kinase domain of VEGFR

  • e.g. Sorafenib, Sunitinib
  • Some level of specificity - because GFs share ATP binding sites, these SMIs can block the ATP binding site but there is overlap with other kinases = multi-kinase inhibitors
• How does thalidomide (+analogues) target angiogenesis?

  • Inhibit phosphorylation of AKT (PKB) which is crucial for downstream signalling of GFs (VEGF, FGF-2 etc.)
  • Leads to inhibition of cell proliferation/blood vessel formation
  • Dangerous during embryogenesis
• What is metastasis?

  Metastasis is the spread of cancer cells from the primary tumor via. the blood and lymphatic system to other parts of the body (secondary site).
• What happens when blood vessels are near the tumour mass?

  Increases the likelihood that cells can break off from the tumour mass, enter the nearby blood vessels, be carried through body to a new site, and recolonise that secondary site
• Describe the stages of lung cancer

  1. Disease has not spread outside of the lung
  2. Disease may spread to lymph nodes or nearby tissues but not in distant parts of the body
  3. Disease can be in >1 lymph nodes or nearby tissues but not in distant parts of the body
  4. Cancer has spread to distant parts of the body

  
• When is cancer considered metastatic?

  Reactivation of tumour cells that were dormant in the body in the same or different tissue are considered metastatic and harder to treat
• What is the tumour microenvironment?

  The surrounding cells, blood vessels, and immune cells that interact with a tumour and make it complex
• Describe the metastatic cascade

  • Primary tumour growth
  • Proliferation
  • Angiogenesis
  • Local invasion, detachment, and intravasation into lymphatics/capillaries
  • Survival in circulation
  • Arrest and extravasation at target/secondary organ
  • Establish pre-metastatic niche - correct GFs and signas develops micrometastases
  • Metastatic colonisation
• What processes occur in the pre-metastatic niche?

  • Death
  • Dormancy
  • Proliferation
• How could we block the recolonisation stage of the metastatic cascade?

  Micrometastases

  • Drive into dormancy - prevent their reactivation
  • Induce cell death before it becomes a secondary growth
• What is the epithelial-mesenchymal transition?

  Cells change their phenotype from epithelial (static, fixed) to mesenchymal (migratory) to acquire motility and invasiveness (break away from the tumour)
• What are features of epithelial cells and mesenchymal cells?

  Epithelial

  • Cytokeratin expression
  • Adherence junctions (E-cadherin)
  • Epithelial polarisation
  • Epithelial markers: E-cadherin, b-catenin

  Mesenchymal

  • Fibroblast-like shape
  • Increased motility and invasiveness
  • Secretion of proteases (MMPs)
  • Mesenchymal markers: N-cadherin, vimentin
• What stage of the metastatic cascade does EMT reverse?

  Extravasation (inside core tissues)

  
  

  Switch back to epithelial phenotype (from mesenchymal)

  Stimulated by both autocrine (tumour) and paracrine (TME) signals like TGFb
• What happens during local invasion?

  Cells undergo EMT and become mobile, secreting matrix metalloproteases (MMPs)
• What happens during intravasation?

  Cells pass paracellularly (intercellular space between the cells) or transcellularly (through the cell itself) through junctions of endothelial cells
• What happens during extravasation?

  Cancer cells exit the bloodstream and enter endothelial cells

  Use selectins (to roll cancer cells along the endothelial surface) and integrins (facilitate the firm adhesion and migration of cancer cells)
• What happens to tumour cells surviving in circulation?

  Circulating tumour cells (CTCs) utilise different mechanisms to survive

  • Remain within the vicinity of other cells
  • Interact with platelets and lymphocytes
  • Possess trophic signals - enough to keep them alive but not to activate
  • If there is not enough GF signalling (to survive), leads to apoptosis
• What is anoikis?

  Anoikis is cell death due to loss of attachment
• How do tumour cells respond to anoikis?

  • Adapt their metabolism to keep ATP and protein productions to protect them
  • Increase survival siganlling via. other pathways
  • Increase expression of pro-survival factors (Bcl-2 family members)
• What are the theories on where cancer spreads?

  1. Random - no particular destination, can go anywhere
  2. First pass organ - recolonisation in the first organ encountered
  3. Seed and soil theory - extravasates to the organ that provides a fertile environment to support growth of tumours cells
• How does the primary tumour prepare the pre-metastatic niche?

  • Provides the right GFs, TME (myeloid-derived suppressor cells and tumour exosomes) within secondary tissues
• Why are dormant tumour cells a problem?

  • Dormant cells arrest tumour growth
  • As they do not rapidly divide, they are no longer susceptible to chemotherapy and targeted therapy
  • Unknown what dormant cells express (for targeting) - need to find a way to target them
• How are matrix metalloprotease inhibitors used to target the metastatic cascade?

  MMPs are key in digesting ECM

  
  

  1st generation = Marimastat

  • Poor efficacy
  • High toxicity
  • Lacked specificity - many MMPs exist
• How are VEGFR/MET (EMT receptor) inhibitors used to target metastatic cascade?

  
  

  Cabozantinib

  • Approved for the treatment of metastatic renal cell carcinoma (mRCC) and hepatocellular carcinoma (HCC)
  • Demonstrated efficacy in inhibiting tumor angiogenesis, reducing metastatic spread, and improving progression-free survival and overall survival in clinical trials