Week 7

• Epithelial cells -> are cells that come from surfaces of your body, such as your skin, blood vessels, urinary tract, or organs. They serve as a barrier between the inside and outside of your body and protect it from harmful agents.

  
• Carcinomas are cancers specific to epithelial cells and result in the disruption of intercellular junctions and alterations in cell polarity.

  
• • hyperplasia - the packing of the cells is irregular and does not line up with other cells
  • dysplasia - a lot of mutation and undetected growth
  • In situ cancer - cancer sitting in one spot
  • invasive cancer - involves epithelial cells departing their normal tissue niche and invading (occupy) other parts of the local tissue or organ.

  
• Epithelial-mesenchymal transition (EMT)

  • Dissolution of cell-cell junction
  • Cytoskeletal rearrangements
  • increased motility
  • Synthesis of extracellular matrix

  
• EMT is a regulated change in the state of an epithelial cell to become a mesenchymal cell. Mesenchymal cells are migratory can develop invasive properties and can remodel (degrade and re-form) the extracellular matrix. EMT can be detected by gain or loss of certain proteins (E.g. LOSS of E-cadherin)

  
• EMT is required for development but also contributes to cancer progression. EMT occurs at specific key times during normal development. In cancer, EMT occurs when it is not supposed to, which can lead to cells becoming migratory and invasive.

  
• EGFR activates many signals and controls many cell outcomes. Ligand binding to EGFR causes the activation of many signals, some sequential, and some parallel

  
• In the MAPK pathway, there are 3 important negative feedback loops.

  • Ras hydrolysis of GTP to GDP inactivates the pathway
  • Mutation in Ras to hydrolyze GTP can cause cancer
  • ERK can phosphorylate MEK to turn it off
  • ERK can also phosphorylate RAF and EGFR to inactivate their kinase ability
  • Phosphorylated EGFR by ERK allows it to be rapidly ubiquitinated.

  
• EGFR can be internalized after ligand binding --> EGFR degradation requires modification of EGFR with ubiquitin.

  
• Western blotting to detect phosphorylated proteins: starts with SDS-PAGE to separate proteins by molecular weight --> proteins are then transferred to the membrane and specific proteins with antibodies.

  • use an antibody that only binds to proteins with specific PTM modification
  • some antibodies bind to a specific protein regardless of PTM.

  
• Immunofluorescence staining and microscopy:

  • conceptually similar to the detection of proteins by antibodies, except labelling is IN CELLS and uses fluorescence as a readout.
  • Once prepared, slides are imaged by fluorescence microscopy.

  
• Blocking (perturbing) function of signalling intermediates to determine what they do

  • use a chemical inhibitor that only blocks the action of one kinase
  • Then, determine if receptor kinase is still able to control cell growth

  
• Another type of blocking

  • Small interfering RNA (siRNA)
  • siRNAs are an alternative to drugs for blocking the function of a specific protein/gene
  • Synthetic siRNA of a sequence complementary to a region of the target gene is delivered to a cell.
  • siRNA is loaded onto a protein complex called RISC (degrade dsRNA)
  • siRNA recognizes and binds target gene mRNA by complementary base-pairing.
  • Target mRNA is then destroyed -> which silences translation of the associated protein product in the cell.

  
• What did they find in the epithelial cancerous cell line for the MAPK and Akt pathway using blocking inhibitors?

  • the presence of EGF matters as there is no Erk, Akt, or EGFR phosphorylation without it
  • in the presence of PD there are more EGFR p-Tyr since Erk is not able to dampen EGFR's ability to autophosphorylate its Tyr residues.
  • In the presence of PD there were no signs of phosphorylated Erk.
  • In the presence of LY there were no signs of phosphorylated Akt.

  
• Inhibition of MAPK enhances the rate of EGFR degradation; Inhibition of Akt does not change EGFR degradation.

  
• An inhibitor (PD) of the MAPK pathway resulted in:

  • inhibition of Erk phosphorylation
  • Faster rate of EGFR degradation after adding ligand (EGF)
  • Higher amount of EGFR post-translationally modified with ubiquitin

  
• The binding of EGF to EGFR causes prostate cancer cells to detach from one another and migrate (EMT). In cells treated with LY, the binding of EGF to EGFR does not cause cells to detach and migrate.

  
• • PD inhibits MEK
  • LY inhibits PI3K
  • siRNA is used to silence SNAIL

  
• • Binding of EGF to EGFR prostate cancer cells to lose expression of the protein E-cadherin
  • In cells treated with LY, the binding of EGF to EGFR is NOT ABLE to cause cells to lose expression of E-cadherin

  
• SNAIL is a strong repressor of the transcription of the E-cadherin gene.
• The binding of EGF to EGFR prostate cancer cells causes an increase in the expression of the protein SNAIL. In cells treated with LY, the binding of EGF to EGFR is NOT ABLE to increase the expression of SNAIL

  
• siRNA targeting of SNAIL mRNA leads to silencing of SNAIL protein. Silencing of SNAIL prevents the loss of E-cadherin that otherwise happens when EGF binds to EGFR

  
• In prostate cancer cells, EGF binding to its receptor (EGFR) leads to the activation of BOTH Ras-Erk signals and PI3K-Akt signals

  • Ras-Erk signals delay or impair the negative feedback regulation that causes EGFR degradation
  • PI3K-Akt signals cause epithelial-mesenchymal transition (EMT) by triggering the expression of SNAIL.

  
• Mesenchymal stem cell (MSC) differentiation is controlled by specific hormones. MSC turns into different cells during embryogenesis

  • This allows for the renewal of different cell types and organs
  • Will turn into different linage based on environmental factors
  • growth factors can stimulate MSC into differentiating towards a specific cell type
  • sometimes the growth factors can be inhibitory toward the specific pathway.

  
• Mesenchymal cell differentiation is controlled by specific hormones and their receptors.

  • EGF stimulates the osteogenesis process --> ligand of EGFR
  • PDGF inhibits the osteogenesis process --> ligand of PDGF-receptor

  
• Western blot found that there are differentials between the proteins created when EGF is used compared to when PDGF is used. this shows that growth factors create differentials.

  
• EGF but not PDGF can convert mesenchymal stem cells to osteoclast

  • cells were treated with colorimetric alkaline phosphatase substrate --> red colour is the reaction between alkaline phosphatase and osteoclast
  • when PDGF is present there is no production of Osteoclast
  • when EGF is present, osteoclast formation occurs.

  
• in mesenchymal stem cells:

  • PDGF binding to its receptor causes activation of PI3K
  • EGF binding to its receptor does NOT cause activation of PI3K
  • this could indicate that PI3K activation inhibits the ERK pathway in some way that restricts osteoclast formation

  
• Wortmannin inhibits PI3K --> inhibition of PI3K makes PDGF have the same effect as EGF --> allows for osteoclast development

  
• inhibiting Ras-Erk blocks osteoclast formation from stem cells in all circumstances ( wether we use EGF or PDGF + wortmannin)

  
• The unique activation of PI3K by PDGF receptor is the cause of the different effects of PDGF and EGF on stem cell differentiation.
• 1. Receptor tyrosine kinases activate many different types of signalling pathways
  2. Receptor tyrosine kinase engages specific pathways for specific outcomes
  3. Receptor tyrosine kinase signaling is context-specific
  4. Receptor tyrosine kinases are unique due to the combination of all signals activated