L2 - Self Sufficiency in Growth Flashcards
Describe the basic paradigm that all growth signalling conforms to
Extracellular growth factor binding to a receptor protein
Triggering an intracellular signalling cascade
Resulting in changes in metabolism/protein function/gene expression
What two main ways is singalling controlled
Phosphorylation
GTP binding
Describe signalling through phosphorylation
Using kinases and phosphatases to add or remove phosphate groups
Can turn on or off in both ways
Describe signalling through GTP binding
Protein is active when GTP is bound and is hydrolysed to turn the proteins off - regulated by GAPs and GEFs
Where are RTKs
In the plasma membrane
Describe how RTKs are activated
Free to move around in the plasma membrane - bump into each other and they dimerise
What does phosphorylation allow
Biding of other proteins
Describe the steps that must occur for Ras to become active
Ligand binding to the RTK Dimerisation and autophosphorylation Phosphorylation allows binding of adaptor proteins with SH2 domains (Grb2) Allows binding of Ras GEFs (SOS) Leads to activation of Ras
What technique was used to determine that Ras had two configurations
Xray crystalography
What determines the confirmational state of Ras
GNMP binding
What are the gatekeeper residues of Ras why are they called this?
Gly12 and Gln61
They allow/fascilitate the binding and hydrolysis of GTP
What is critical to the turning on/off of Ras
The shape change
Alows the interaction with downstream effectors
What is FRET
Fluorescent
Describe the principle that FRET works on
Emission/excitation phenomena
Excite protein A at wavelength x causing emssion at wavelength y
Wavelength y is the excitation wavelength for protein B which then causes the emission of light at wavelength z
This concept can only occur if both A and B are close to each other (within a few nanometers
Describe how Ras signalling may be investigated using FRET
Inject a Ras binding protein labelled with cyan FP and Ras labelled with YFP
When Ras signalling is on two proteins will be close (bound) and FRET will be observed
4 ways that self sufficiency in growth signals may arise
Autocrine signalliung
Ligand independent
GTPase siwtch-off failure
Constituitve downstream signalling
What is oncogene adiction
A consequence of self-sufficiency in cell signalling where cells become addicted to a signalling pathway
Mutation and selection drive
Genetic streamlining
Describe the concept of genetic streamlining
Whereby cells under a selection pressure may undergo epigenetic silencing of a single pathway
What is a concequence of genetic streamlining
One pathway becomes dominant and the signal strength goes up
This can be seen through increased expression of a component of the pathway - usually the receptor
What is the consequence of increased levels of the receptor
Cells require a greater signal strength since the number of receptors they express is increased
More receptors = more receptors must be occupied for 100% signal saturation
What is the implication on treamtment of oncogene addiction
For targetted therapies have to target the dominant pathway
There is no effect on targetting pathways that arent the domiannt one
Give an example of a gene that is observed in oncogene addiction
HER 2
Is an RTK which requires ligand mediated dimerisation and subsequent autophosphorylation
Upregulated in a subset of breast cancers - oncogenic addiction
What was the drug used to treat HEr2 OE breast cancer
Trastusamab
Another name for Trastusamab
Herceptin
What is a Kaplan Meirer Plot
Time against survival - of a patient or a model system
Describe the Kaplan Meirer plot for treating with herceptin
Cells with low and high levels of HER2 do quite well when treated with herceptin
Cells with intermediate levels dont respond so well
Paper from this lecture
Self suff in growth signals
Neuregulin-1-mediated autocrine signalling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers`
What are HKI2727 and lapaptinib
Specific inhibtors for the HER1 and HEr2 tyrosine kinase
They are UNABLE to distinguish between the two so inhibit them both eqaully
What is Erlotinib
Specific inhibotr of HER1
Describe the methods used in the Neuregulin-1-mediated autocrine signalling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers paper
Measure cell growth in 72 different cell lines with and without the various drugs (for HER2 kinase inhibition)
Cell growth was measured using a dye which was quantifiable
What did they initially see
Some failed to respond and others are incredibly sensitive
What is neruregulin 1
Ligand for Her1
In the journal paper how was cell growth measured
Using an assay whereby the strength of colour of the dye was dependent on the levels of cell growth
Why was their unexplained sensitivity in some of the cells
Becuase there were no mutations in either the EGF receptor or overexpression of HER2
30 cell lines dispayed unexplained sensitivity
What was the next step once the 30 cell lines with unexplained sensitivity had been identified
Put into 3 categories and measure cell viability against concentration of lapatinib
What were the three categories
Sensitive
Moderately sensitive
Refractory
Describe what was seen when plotting viability against the concentration of drug used
Viabile at low concentrations
Variability of where inhibition seen - could be as low as 0.1 um or as high as 10um
What was the next step in determining why some cells were incredibly sensitive and others not
Using immunoblotting to look at all signalling pathways
What were the results of looking at multiple signalling pathways
Found that in sensitive cells lots of pHER3 relative to the ammount of HER3
In cells that werent sensitive there were still high levels of HER3 but not the phosphorylated version
Sensitivity to lapatinib correlates with
Phosphorylated HER3
What is the action of lapatinib
Her2 inhibitor
Once the correlation between lapatinib sensitivty and pHER3 levels was made what was the next step
Describe how they did this
Looking at expression of the ligand
Detecting if the cells are making the ligand or if the ligand is present in the media suggesting that it is being secreted
Describe the results of looking at the expresison of NRG1
Nearly all lapatinib sensitive cells express NRG1 - suggests the cancer cells are making this ligand
In all the cultures of lapatinib sensitive cells NRG1 was seen - suggests that the cells are secreting this ligand
Was looking at NRG1 levels the final step?
What question still needed to be addressed
No
Needed to confirm that cell viability in these sensitive cells is a function of whether they express NRG1
How was the question that cell viability in these sensitive cells is a function of whether they express NRG1 tested.
Describe the results
Using a genetic knockdown of NRG1 and then exogenous knockback of NRG1
When NRG1 is knocked down there is a significant decrease in cell viability
When NRG1 is knocked back see a rescue - cells begin to grow again
Summarise the findings of Neuregulin-1-mediated autocrine signalling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers`
NRG1 is made and secreted by these cells which then binds to Her3
Autocrine signalling
But if lapatinib inhibits Her2 then how does it impact on the function of the Her3 pathway
Her3 forms heterodimers with Her2
Describe the proof of concept from the NRG paper
If you have a model tumour expressing Her2 and Her3 then it should be able to be treated ususing lapatinib
See a significant decrease in the rate of growth when lapatinib was added
This upregulation of NRG1 is known as
AUTOCRINE SIGNALLING
NSCLC and neuroglioblastoma both commonly have a _____ of function mutation in what gene
GAIN IN EGFR
Describe the effect of a gain of function mutation in EGFR
Loss of the EC domains meas that the receptors may dimerise independent of the ligand
The gain of function in EGFR is known as
LIGAND INDEPENDENT SIGNALLING
Describe the EC domain of the EGFR
4 domains within itself
Describe the conformation of the EGFR in the absence of the ligand
EC domain folded back and tethered
Dimerisation domain is hidden in the monomeric form
Describe how dimerisation of the EGFR occurs
Arm rotates upwards and is held in place by the ligand
Dimerises to a second EGFR
What were the results when exposing the EGFR to small angle xray scattering and single molecule cryo EM
Can see the receptor exists in two confirgurations
DImerisation requires the open configuartion
What is STORM
Stochastic optical reconstruction microscotpy
Describe how storm was used to investigate the EGFR
EGFR was tagged in two colours using a fluro nano particle - where see two dots together = DIMER
Can see when there is no ligand there is no association of the green and purple dots and no dimerisation
When ligand added the dots come together - LIGAND DRIVES DIMERISATION
What is seen when we use STORM on common EGF mutants
Dimerisation occurs in the absence of the ligand
What is the function of the interaction domains
Keep the dimerisation domain hidden between domain 4 and 2
Describe a mutation of the EGFR and an effect this had
Single point mutation - place where the arms isnt held in place - dimerisation domain not hidden and can occur even without the presence of a lignad
How many isoforms of Ras
3
h K N
In what percentage of tumours is Ras mutated
75%
The fact Ras is mutated in 75% of cancers implies
Ras is a major drive mutation
What is the experimental paradigm used form confirming oncogenes
Transfect normal mouse fibroblasts with the oncogene
Inject transformed cells into mouse
If its an oncogene then will see the formation of a tumour
What is the difference between normal and oncogenic ras
At position 12
Glycine –> Valine
What is significant about the region different in normal and onco ras
Position 12
The position where GTP hydrolysis has to occur
How did we discover that mutant Ras was unable to hydrolyse gtP
Analysis of bound nucleotides by chromatograpy
Incubate with radio-guanine
In the WT over time see increase in GDP levels
In the mutant no increase in GDP so GTP not being hydrolysed
How does Ras promote growth
Raf –> Mek –> ERk (Map Kinase)
How does Ras supress death
PI3K –> PIP3 –> Akt –> Supression of apoptosis
How does Ras promote cell migration
Production of lammelipodia and filopodia
What are the three main funcitions of Ras
Promote growth
Supress death
Promote migreation
Where do the mutations occur in Ras
What is the impact
GTP hydrolysis - pos 12, 13 and 61
Effector loops - 30-44 and 55-65
What is different about Ras in different tissues
Different forms of Ras are expressed in different tissues
What can be seen about the likelyhood of mutations at a postion in different Ras isofroms
Diffrent Ras isoforms have a different likelihood of being mutated at the various positions
Give an explanation for the different mutational profiles seen in different tissues and isoforms
In lung cancer K ras is frequently mutated at position 12
Glycine –> Cystine
Benzopyrene is a derivative of guanine and forms guanine benzopyrene
This is bulky and requires the mismatch repair pathways to remove
Mismatch repair pathways mistake guanine benzopyrene for a T
So at position 12 GGT goes to TGT
What happens to the K Ras G12C mutation when people stop smoking
This incidence progressively declines to 0
What can G12C be used as
A diganostic marker
What is the evidence for tissue specific different in exposure to individual tobacco smoke mutagens
The C12G mutation is far less abundant in pancreatic and colon cancer which are also affected by smoking
Where is N ras frequently mutated
At position 61
Gln –> Leu
What is the reason for high frequency of mutations in N ras at position 61
UV light is capable of inducing pyrimidine dimers A-T
Where do mutations in Ras mainly occur
In the regions associated with GTP hydrolysis or effector loops
What is the effect of crossing a floxxed Val12 Ras mutant mouse with a mouse carrying Cre recombinase under the control of a tissue specific promoter
Expression of mutant Ras in the Lungs
When expression of Val12 mutant Ras is driven in the lungs what is seen
Can see senescence markers in adeonmas
Expression of oncogenic Ras actually causes senescence
What is the sweet spot hypothesis
The idea that there is a continum of behaviours
Regulated growth
Excessive growth
Senescence
When cells are excessively growing this is when tumours are likely to be very dangerous
SWEET SPOT HYPOTHESIS
Where would a WT Ras homo be
In the regulated growth
SWEET SPOT HYPOTHESIS
Where would a Ras mut het be
In the excessive growth - dangerous
SWEET SPOT HYPOTHESIS
Where would ras mut homo be
In the senescence - overdose of the oncogene
What occurs to the SWEET SPOT HYPOTHESIS when cells enter true neoplasia
Bar changes
Excessive signalling now longer causes senescence
What is constituitve downstream activation
Can acquire mutations at points in the pathway which turn on that component without the need for activation from a higher source
What is Raf
Protein kinase with a Ras biding domain
What are the different domains within the Ras kinase domain
ATP binding domain
Activation loop
What happens to Raf when it binds to Ras
Causes swinging out of the way to turn the kinase domain on
Describe Raf in the absence of Ras binding
Ras binding domain prevents the activation of the kinase domain
How might the regulation of Raf go wrong
Mutation V600E prevents the closing of the strucutre and ATP is always bound and the kinase domain is always active
What cancers is the V600E mutation seen in
Melanoma Colorectal Thyroid Glioblastoma NSCLC
How may V600E Raf mutants
Give examples of two compounds
Using raf inhibitros
Babrafenid and vemurafenib
Describe why the acquistion of resitance to Raf happens quickly
Raf tends to aligomerise and Ras interferences with itself to stop signalling Low levels of the inhibotr leads to activation and very high levels of the inhibitor is required
Describe the concept of oncogene overdose of BRAF
BRAF mutated melanoma - regression in treament with vemurafenib (Raf inhibitor)
But sustained treatment leads to the emergence of lethal drug resistance disease due to BRAF V600E
BUT THEY ARE ALSO DRUG DEPENDENT FOR THEIR PROLIFERATION
Describe how cells with mutant BRAF adopt resistance
Upregulation of BRAF - so there are always dimers without the drug bound to them
Describe how you would treat cancers with oncogenic addiction
Cancer ill regress - then have cancer that emerges - then have to take it away again
THIS IS KNOWN AS ONCOGENE OVERDOES INTERMITTENT THERAPY
