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BMS379 - Cancer Biology > L2 - Self Sufficiency in Growth > Flashcards

L2 - Self Sufficiency in Growth Flashcards

(102 cards)

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1
Q

Describe the basic paradigm that all growth signalling conforms to

A

Extracellular growth factor binding to a receptor protein
Triggering an intracellular signalling cascade
Resulting in changes in metabolism/protein function/gene expression

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2
Q

What two main ways is singalling controlled

A

Phosphorylation

GTP binding

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3
Q

Describe signalling through phosphorylation

A

Using kinases and phosphatases to add or remove phosphate groups
Can turn on or off in both ways

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4
Q

Describe signalling through GTP binding

A

Protein is active when GTP is bound and is hydrolysed to turn the proteins off - regulated by GAPs and GEFs

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5
Q

Where are RTKs

A

In the plasma membrane

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6
Q

Describe how RTKs are activated

A

Free to move around in the plasma membrane - bump into each other and they dimerise

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7
Q

What does phosphorylation allow

A

Biding of other proteins

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8
Q

Describe the steps that must occur for Ras to become active

A 
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
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9
Q

What technique was used to determine that Ras had two configurations

A

Xray crystalography

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10
Q

What determines the confirmational state of Ras

A

GNMP binding

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11
Q

What are the gatekeeper residues of Ras why are they called this?

A

Gly12 and Gln61

They allow/fascilitate the binding and hydrolysis of GTP

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12
Q

What is critical to the turning on/off of Ras

A

The shape change

Alows the interaction with downstream effectors

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13
Q

What is FRET

A

Fluorescent

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14
Q

Describe the principle that FRET works on

A

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

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15
Q

Describe how Ras signalling may be investigated using FRET

A

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

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16
Q

4 ways that self sufficiency in growth signals may arise

A

Autocrine signalliung
Ligand independent
GTPase siwtch-off failure
Constituitve downstream signalling

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17
Q

What is oncogene adiction

A

A consequence of self-sufficiency in cell signalling where cells become addicted to a signalling pathway

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18
Q

Mutation and selection drive

A

Genetic streamlining

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19
Q

Describe the concept of genetic streamlining

A

Whereby cells under a selection pressure may undergo epigenetic silencing of a single pathway

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20
Q

What is a concequence of genetic streamlining

A

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

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21
Q

What is the consequence of increased levels of the receptor

A

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

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22
Q

What is the implication on treamtment of oncogene addiction

A

For targetted therapies have to target the dominant pathway

There is no effect on targetting pathways that arent the domiannt one

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23
Q

Give an example of a gene that is observed in oncogene addiction

A

HER 2
Is an RTK which requires ligand mediated dimerisation and subsequent autophosphorylation

Upregulated in a subset of breast cancers - oncogenic addiction

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24
Q

What was the drug used to treat HEr2 OE breast cancer

A

Trastusamab

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25
Another name for Trastusamab
Herceptin
26
What is a Kaplan Meirer Plot
Time against survival - of a patient or a model system
27
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
28
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`
29
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
30
What is Erlotinib
Specific inhibotr of HER1
31
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
32
What did they initially see
Some failed to respond and others are incredibly sensitive
33
What is neruregulin 1
Ligand for Her1
34
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
35
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
36
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
37
What were the three categories
Sensitive Moderately sensitive Refractory
38
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
39
What was the next step in determining why some cells were incredibly sensitive and others not
Using immunoblotting to look at all signalling pathways
40
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
41
Sensitivity to lapatinib correlates with
Phosphorylated HER3
42
What is the action of lapatinib
Her2 inhibitor
43
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
44
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
45
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
46
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
47
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
48
But if lapatinib inhibits Her2 then how does it impact on the function of the Her3 pathway
Her3 forms heterodimers with Her2
49
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
50
This upregulation of NRG1 is known as
AUTOCRINE SIGNALLING
51
NSCLC and neuroglioblastoma both commonly have a _____ of function mutation in what gene
GAIN IN EGFR
52
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
53
The gain of function in EGFR is known as
LIGAND INDEPENDENT SIGNALLING
54
Describe the EC domain of the EGFR
4 domains within itself
55
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
56
Describe how dimerisation of the EGFR occurs
Arm rotates upwards and is held in place by the ligand | Dimerises to a second EGFR
57
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
58
What is STORM
Stochastic optical reconstruction microscotpy
59
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
60
What is seen when we use STORM on common EGF mutants
Dimerisation occurs in the absence of the ligand
61
What is the function of the interaction domains
Keep the dimerisation domain hidden between domain 4 and 2
62
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
63
How many isoforms of Ras
3 h K N
64
In what percentage of tumours is Ras mutated
75%
65
The fact Ras is mutated in 75% of cancers implies
Ras is a major drive mutation
66
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
67
What is the difference between normal and oncogenic ras
At position 12 Glycine --> Valine
68
What is significant about the region different in normal and onco ras
Position 12 | The position where GTP hydrolysis has to occur
69
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
70
How does Ras promote growth
Raf --> Mek --> ERk (Map Kinase)
71
How does Ras supress death
PI3K --> PIP3 --> Akt --> Supression of apoptosis
72
How does Ras promote cell migration
Production of lammelipodia and filopodia
73
What are the three main funcitions of Ras
Promote growth Supress death Promote migreation
74
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
75
What is different about Ras in different tissues
Different forms of Ras are expressed in different tissues
76
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
77
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
78
What happens to the K Ras G12C mutation when people stop smoking
This incidence progressively declines to 0
79
What can G12C be used as
A diganostic marker
80
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
81
Where is N ras frequently mutated
At position 61 Gln --> Leu
82
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
83
Where do mutations in Ras mainly occur
In the regions associated with GTP hydrolysis or effector loops
84
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
85
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
86
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
87
SWEET SPOT HYPOTHESIS Where would a WT Ras homo be
In the regulated growth
88
SWEET SPOT HYPOTHESIS Where would a Ras mut het be
In the excessive growth - dangerous
89
SWEET SPOT HYPOTHESIS Where would ras mut homo be
In the senescence - overdose of the oncogene
90
What occurs to the SWEET SPOT HYPOTHESIS when cells enter true neoplasia
Bar changes | Excessive signalling now longer causes senescence
91
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
92
What is Raf
Protein kinase with a Ras biding domain
93
What are the different domains within the Ras kinase domain
ATP binding domain | Activation loop
94
What happens to Raf when it binds to Ras
Causes swinging out of the way to turn the kinase domain on
95
Describe Raf in the absence of Ras binding
Ras binding domain prevents the activation of the kinase domain
96
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
97
What cancers is the V600E mutation seen in
``` Melanoma Colorectal Thyroid Glioblastoma NSCLC ```
98
How may V600E Raf mutants Give examples of two compounds
Using raf inhibitros Babrafenid and vemurafenib
99
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
100
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
101
Describe how cells with mutant BRAF adopt resistance
Upregulation of BRAF - so there are always dimers without the drug bound to them
102
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

BMS379 - Cancer Biology (11 decks)

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