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

L2 - Self Sufficiency in Growth Flashcards

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AP® Biology flashcards Biology 101 flashcards
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
Q

Another name for Trastusamab

A

Herceptin

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26
Q

What is a Kaplan Meirer Plot

A

Time against survival - of a patient or a model system

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27
Q

Describe the Kaplan Meirer plot for treating with herceptin

A

Cells with low and high levels of HER2 do quite well when treated with herceptin

Cells with intermediate levels dont respond so well

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28
Q

Paper from this lecture

Self suff in growth signals

A

Neuregulin-1-mediated autocrine signalling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers`

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29
Q

What are HKI2727 and lapaptinib

A

Specific inhibtors for the HER1 and HEr2 tyrosine kinase

They are UNABLE to distinguish between the two so inhibit them both eqaully

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30
Q

What is Erlotinib

A

Specific inhibotr of HER1

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31
Q

Describe the methods used in the Neuregulin-1-mediated autocrine signalling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers paper

A

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

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32
Q

What did they initially see

A

Some failed to respond and others are incredibly sensitive

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33
Q

What is neruregulin 1

A

Ligand for Her1

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34
Q

In the journal paper how was cell growth measured

A

Using an assay whereby the strength of colour of the dye was dependent on the levels of cell growth

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35
Q

Why was their unexplained sensitivity in some of the cells

A

Becuase there were no mutations in either the EGF receptor or overexpression of HER2
30 cell lines dispayed unexplained sensitivity

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36
Q

What was the next step once the 30 cell lines with unexplained sensitivity had been identified

A

Put into 3 categories and measure cell viability against concentration of lapatinib

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37
Q

What were the three categories

A

Sensitive
Moderately sensitive
Refractory

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38
Q

Describe what was seen when plotting viability against the concentration of drug used

A

Viabile at low concentrations

Variability of where inhibition seen - could be as low as 0.1 um or as high as 10um

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39
Q

What was the next step in determining why some cells were incredibly sensitive and others not

A

Using immunoblotting to look at all signalling pathways

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40
Q

What were the results of looking at multiple signalling pathways

A

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

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41
Q

Sensitivity to lapatinib correlates with

A

Phosphorylated HER3

42
Q

What is the action of lapatinib

A

Her2 inhibitor

43
Q

Once the correlation between lapatinib sensitivty and pHER3 levels was made what was the next step

Describe how they did this

A

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
Q

Describe the results of looking at the expresison of NRG1

A

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
Q

Was looking at NRG1 levels the final step?

What question still needed to be addressed

A

No

Needed to confirm that cell viability in these sensitive cells is a function of whether they express NRG1

46
Q

How was the question that cell viability in these sensitive cells is a function of whether they express NRG1 tested.

Describe the results

A

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
Q

Summarise the findings of Neuregulin-1-mediated autocrine signalling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers`

A

NRG1 is made and secreted by these cells which then binds to Her3

Autocrine signalling

48
Q

But if lapatinib inhibits Her2 then how does it impact on the function of the Her3 pathway

A

Her3 forms heterodimers with Her2

49
Q

Describe the proof of concept from the NRG paper

A

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
Q

This upregulation of NRG1 is known as

A

AUTOCRINE SIGNALLING

51
Q

NSCLC and neuroglioblastoma both commonly have a _____ of function mutation in what gene

A

GAIN IN EGFR

52
Q

Describe the effect of a gain of function mutation in EGFR

A

Loss of the EC domains meas that the receptors may dimerise independent of the ligand

53
Q

The gain of function in EGFR is known as

A

LIGAND INDEPENDENT SIGNALLING

54
Q

Describe the EC domain of the EGFR

A

4 domains within itself

55
Q

Describe the conformation of the EGFR in the absence of the ligand

A

EC domain folded back and tethered

Dimerisation domain is hidden in the monomeric form

56
Q

Describe how dimerisation of the EGFR occurs

A

Arm rotates upwards and is held in place by the ligand

Dimerises to a second EGFR

57
Q

What were the results when exposing the EGFR to small angle xray scattering and single molecule cryo EM

A

Can see the receptor exists in two confirgurations

DImerisation requires the open configuartion

58
Q

What is STORM

A

Stochastic optical reconstruction microscotpy

59
Q

Describe how storm was used to investigate the EGFR

A

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
Q

What is seen when we use STORM on common EGF mutants

A

Dimerisation occurs in the absence of the ligand

61
Q

What is the function of the interaction domains

A

Keep the dimerisation domain hidden between domain 4 and 2

62
Q

Describe a mutation of the EGFR and an effect this had

A

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
Q

How many isoforms of Ras

A

3

h K N

64
Q

In what percentage of tumours is Ras mutated

A

75%

65
Q

The fact Ras is mutated in 75% of cancers implies

A

Ras is a major drive mutation

66
Q

What is the experimental paradigm used form confirming oncogenes

A

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
Q

What is the difference between normal and oncogenic ras

A

At position 12

Glycine –> Valine

68
Q

What is significant about the region different in normal and onco ras

A

Position 12

The position where GTP hydrolysis has to occur

69
Q

How did we discover that mutant Ras was unable to hydrolyse gtP

A

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
Q

How does Ras promote growth

A

Raf –> Mek –> ERk (Map Kinase)

71
Q

How does Ras supress death

A

PI3K –> PIP3 –> Akt –> Supression of apoptosis

72
Q

How does Ras promote cell migration

A

Production of lammelipodia and filopodia

73
Q

What are the three main funcitions of Ras

A

Promote growth
Supress death
Promote migreation

74
Q

Where do the mutations occur in Ras

What is the impact

A

GTP hydrolysis - pos 12, 13 and 61

Effector loops - 30-44 and 55-65

75
Q

What is different about Ras in different tissues

A

Different forms of Ras are expressed in different tissues

76
Q

What can be seen about the likelyhood of mutations at a postion in different Ras isofroms

A

Diffrent Ras isoforms have a different likelihood of being mutated at the various positions

77
Q

Give an explanation for the different mutational profiles seen in different tissues and isoforms

A

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
Q

What happens to the K Ras G12C mutation when people stop smoking

A

This incidence progressively declines to 0

79
Q

What can G12C be used as

A

A diganostic marker

80
Q

What is the evidence for tissue specific different in exposure to individual tobacco smoke mutagens

A

The C12G mutation is far less abundant in pancreatic and colon cancer which are also affected by smoking

81
Q

Where is N ras frequently mutated

A

At position 61

Gln –> Leu

82
Q

What is the reason for high frequency of mutations in N ras at position 61

A

UV light is capable of inducing pyrimidine dimers A-T

83
Q

Where do mutations in Ras mainly occur

A

In the regions associated with GTP hydrolysis or effector loops

84
Q

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

A

Expression of mutant Ras in the Lungs

85
Q

When expression of Val12 mutant Ras is driven in the lungs what is seen

A

Can see senescence markers in adeonmas

Expression of oncogenic Ras actually causes senescence

86
Q

What is the sweet spot hypothesis

A

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
Q

SWEET SPOT HYPOTHESIS

Where would a WT Ras homo be

A

In the regulated growth

88
Q

SWEET SPOT HYPOTHESIS

Where would a Ras mut het be

A

In the excessive growth - dangerous

89
Q

SWEET SPOT HYPOTHESIS

Where would ras mut homo be

A

In the senescence - overdose of the oncogene

90
Q

What occurs to the SWEET SPOT HYPOTHESIS when cells enter true neoplasia

A

Bar changes

Excessive signalling now longer causes senescence

91
Q

What is constituitve downstream activation

A

Can acquire mutations at points in the pathway which turn on that component without the need for activation from a higher source

92
Q

What is Raf

A

Protein kinase with a Ras biding domain

93
Q

What are the different domains within the Ras kinase domain

A

ATP binding domain

Activation loop

94
Q

What happens to Raf when it binds to Ras

A

Causes swinging out of the way to turn the kinase domain on

95
Q

Describe Raf in the absence of Ras binding

A

Ras binding domain prevents the activation of the kinase domain

96
Q

How might the regulation of Raf go wrong

A

Mutation V600E prevents the closing of the strucutre and ATP is always bound and the kinase domain is always active

97
Q

What cancers is the V600E mutation seen in

A 
Melanoma 
Colorectal 
Thyroid 
Glioblastoma 
NSCLC
98
Q

How may V600E Raf mutants

Give examples of two compounds

A

Using raf inhibitros

Babrafenid and vemurafenib

99
Q

Describe why the acquistion of resitance to Raf happens quickly

A

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
Q

Describe the concept of oncogene overdose of BRAF

A

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
Q

Describe how cells with mutant BRAF adopt resistance

A

Upregulation of BRAF - so there are always dimers without the drug bound to them

102
Q

Describe how you would treat cancers with oncogenic addiction

A

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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