Gan et al. Paper Investigation

• hypothesis -> prostate cancer cells use 2 signalling pathways (MAPK and Akt) to specifically lead to EMT and control feedback regulation

  objectives -> use inhibitors of each pathway and determine the effect on EGFR negative feedback (ubiquitination and degradation) and EMT progression
• method 1: western blotting to detect phosphorylated proteins

  • used antibodies that
  • only binds to proteins with specific PTM
  • only binds to specific protein, PTM doesn't matter
  • only binds to any protein with specific phosphorylated residue like serine or tyrosine

  
• method 2: immunofluorescence staining and microscopy

  • proteins are detected with primary and secondary antibodies but the labelling occurs IN CELLS and uses fluorescence as a readout rather than running on SDS PAGE (western blot)

  
• method 3: blocking/perturbing function of signalling intermediates to determine its function

  • used small interfering RNA (siRNA) which are short sequences complementary to a specific mRNA and turns it into a partial duplex -> recognized by RISC assembly and targeted for degradation -> function of protein/gene is blocked/silenced because translation can't occur
  • this is used because deleting genes from the genome takes a longer time

  
• overall experimental plan:

  • use inhibitors of each pathway
  • PD -> Mek inhibitor, indicated by no pErk
  • LY -> PI3K inhibitor, indicated by no pAkt
  • siRNA silences SNAIL

  determine global impact

  • EMT induction -> cell morphology changes and E-cadherin expression (only in epithelial cells)
  • rate of EGFR degradation
  • SNAIL expression -> repressor TF for E-cadherin

  
• results in the CANCEROUS cell line

  • the non phosphorylated version of proteins involved (EGFR, Erk, Akt) are present all the time but their phosphorylated versions are present when EGF is added
  • when PD (Mek) and LY (PI3K) inhibitors are added, the phosphorylated versions are absent when EGF is added
  • BUT there is increased phosphorylation of EGFR when PD is added to the cancer cell line (the non inhibited cancer cell line is preventing negative feedback by having pErk phosphorylate Thr residues to dampen degradation)
  • PD is mimicking the effects of a healthy cell!

  
• inhibition of MAPK enhances rate of EGFR degradation, inhibition of Akt does not change rate of EGFR degradation

  
• cells treated with PD (Mek inhibitor) the binding of EGF to EGFR causes MORE ubiquitination of EGFR

  
• summary:

  • PD inhibitor of MAPK pathway results in
  • inhibition of Erk phosphorylation
  • faster rate of EGFR degradation after adding EGF
  • higher amount of EGFR post translationally modified with ubiquitin protein
  • all this ^ causes the cancer cell line to behave like a normal healthy cell when PD is added
• NORMALLY, binding of EGF to EGFR causes cancer cells to detach and migrate (EMT)

  BUT when cells are treated with LY (PI3K inhibitor) -> binding of EGF to EGFR does NOT cause cell detachment and migration

  • thus adding LY (inhibit Akt pathway) -> prevent EMT pathway from occurring

  
• • cancer cells -> EGF binding to EGFR -> cells lose E-cadherin expression which requires PI3K/Akt which indicates that EMT is happening
  • cancer cells treated with LY (PI3K inhibitor) -> EGF binding to EGFR does NOT cause cancer cells to lose E-cadherin expression

  
• concept reminder: EGF binding to EGFR = induce migration, detachment, proliferation = EMT -> requires Akt pathway and must lose E-cadherin (in epithelial cells tight junctions) to achieve this
• SNAIL is a repressive TF for E-cadherin

  • increase in SNAIL TF -> binds E-cadherin gene and represses its transcription, it's a negative regulator
  • MORE SNAIL -> LESS E-cadherin
• • EGF binding to EGFR -> increase in SNAIL expression, requiring action of Akt
  • LY inhibitor -> EGF binding to EGFR cannot increase SNAIL expression because Akt is not functional

  
• EMT requires SNAIL upon EGF binding to EGFR to ensure loss of E-cadherin

  • this was found when siRNA was used to silence SNAIL mRNA

  
• summary for Akt pathway:

  • in cancer cells, EGF binding to EGFR leads to activation of both MAPK and Akt signal pathways
  • MAPK signals in cancer cell lines can delay/impair negative feedback regulation that would cause EGFR degradation
  • Akt signals in cancer cell lines can cause EMT by triggering SNAIL expression (downregulates E-cadherin, loss of tight junction)

  
• normally, EGF binds EGFR which makes the tyrosine kinase activity turn on and do multiple things including recruiting factors to cause ubiquitination and degradation BUT in cancer cells the pErk still phosphorylates EGFR but on its threonine residue to the reduce tyrosine kinase activity
• the addition of PD inhibitor increases degradation BECAUSE

  • if Erk isn't phosphorylated by Mek -> it can't phosphorylate the threonine residue on EGFR (which is what dampens the EGFR phosphorylated tyrosine kinase activity that causes degradation) -> so since there is no dampening of the tyrosine kinase activity -> degradation of EGFR is increased

  
• phosphorylation of EGFR on

  • SPECIFIC Thr residue -> dampen ubiquitination
  • Tyr residue -> enhance ubiquitination

  when pErk phosphorylates specific Thr residue on EGFR (inhibits kinase ability to auto phosphorylate Tyr (which would increase ubiquitination) -> less ubiquitination
• pErk only phosphorylates Ser/Thr residues, in the cancer cell line, pErk will phosphorylate the Thr residue which inhibits EGFR auto phosphorylation activity (normally this auto phosphorylation is on tyrosine residues which causes ubiquitination)