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CAN > Week 2 > Flashcards

Week 2 Flashcards

1
Q

Intracellular signaling

A

Set of linked biochemical events connecting a stimulus with a response

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2
Q

Communication between cells controls cell behaviour and survival

A

Differnation signals ->Quiescence differentiation
Growth factors-> Proliferation
Death signals->Cell death (apoptosis)

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3
Q

Signal molecules can bind intracellular or cell surface receptors

A

Hydrophilic signals: these bind to the receptors on the cell surface membrane
Small hydrophobic signals: that can bypass the cell membrane and go directly into the cell and bind to a receptor within the cells eg hormones

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4
Q

Receptor signalling cascades and intracellular proteins

A

A signal binds to a receptor
This activates lots of downstream proteins which then:
-activate multiple downstream factors to allow for multiple different effects
-acts as signal amplifiers to allow for greater effects
One of the ultimate effects can be gene expression, metabolism or an effect on cell cytoskeleton

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5
Q

Phosphorylation and cell signalling

A

Phosphorylation is mediated by kinases which act to catalyse phosphorylation
Phosphatases removes phosphorylation to return the protein back to normal
Can occur on 3 residues: threonine, tyrosine, serine residues
Different kinases have different specificity for what residue they phosphorylate
Phosphorylation is an example of post transcriptional modification of the protein
Phosphorylation can act as a binding site on the protein for phospho reader proteins, it might increase/supres the activity of the protein

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6
Q

Growth factors and receptor tyrosine kinase

A

Platelet derived growth factor PDGF
Epidermal growth factor EGF
Insulin
Insulin like growth factor IGF
Transforming growth factor TGFa and B
Nerve growth factor NGF
Vascular endothelial growth factor VEGF
Macrophage-colony-stimulating factor MCSF
Ephrins

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7
Q

Receptor tyrosine kinases: an important class of cell surface receptor

A

Membrane bound receptors
Phosphorylate things on tyrosine residues
All have a kinase domain and extracellular domain
When in an inactive state they exist as monomers
When in active state they exist as dimers
Most are homodimers (identical monomer) but some can heterodimerise

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8
Q

Growth factor binding to tyrosine kinases

A

Causes:
-conformational change
-dimerisation
-activation of tyrosine kinase activity of the cytoplasmic domain
-transphosphorylation- tyrosine kinase on left phosphorylated one on right vice versa

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9
Q

Growth factors and receptor tyrosine kinases II

A

After transphosphorylation you additionally get autophosphorylation of the RTK to further activate tyrosine kinases activity
Various cytoplasmic proteins bind to the phosphorylated receptor
This allows for 2 things to happen:
-some recruited proteins become activated by tyrosine phosphorylation
-facilitates interactions between recruited proteins or with other factors localised at the plasma membrane
Theres considerable overlap in cytoplasmic signalling pathways activated by different growth factor receptors (eg PDGF and FGF receptors)

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10
Q

Summary of MAP (mitogen activated protein) kinase pathway

A

One of molecules that can activate MAPK pathway is EGF
It binds to the EGF receptor which is then activated and can recruit downstream proteins such as GRB2
GRB2 acts to activate other downstream proteins such as RAS
RAS becomes activated and binds to other factors which results in a downstream cascade allowing for the activation of other kinases -> impact on cell growth
I.e MAPK pathways drive cell growth and one of the signals that drive it is EGF

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11
Q

Grb2 and Sos

A

Grb2: growth factor receptor bound protein 2, contains SH2 domain that binds P-Tyr residues on RTKs
Grb2 binds to Sos
Assembly of receptor-Grb2-Sos complex enables recruitment of RAS
If you dont have Sos no recruitment of RAS
If you dont have phosphorylation of RTK then don’t recruit RAS either or Sos

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12
Q

The RAS family

A

3 Ras genes: h-Ras, k-Ras, n-Ras
Encode “G-proteins” (GTP- coupled signal transducing protein)
Contains small lipid group that attaches Ras to membrane
RAS is most commonly mutated oncogene, implicated in 20-30% human cancers
Function:
-in inactive form binds GDP
-if you get a signal it exchanges the GDP to GTP and becomes active
-it can then pass the signal downstream

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13
Q

The RAS-GDP/RAS-GTP cycle

A

Inactive RAS binds to GDP
When we have a growth signal eg EGF it results in RAS exchanging GDP to GTP, it does this with help of other proteins called guanine nucleotide exchange factors GEFS which help speed up the exchange
This results in activation of RAS and the signal is then transduced allowing for an effect eg gene expression
Then RAS turns itself off
-able to do this because it has a GTPase which hydrolyses GTP for GDP, this is stimulated by other proteins called GTPase-activating proteins
A lot of mutations in RAS stop its ability to inactivate itself- cancer

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14
Q

RAS and Sos

A

Important thing about the Grb/SOS receptor is that they act as a GEF (guanine nucleotide exchange factor) for RAS so they ie turn RAS on and so we get signal transduction downstream

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15
Q

RAS in signal transduction

A

Activated RAS binds another protein called RAF
RAF allows the cell to activate downstream kinases such as MEK kinase via phosphorylation
MEK then goes on to phosphorylate other signals eg ERK
Activation of these 2 sets of kinases MEK and ERK ultimately lead to activation of lots of transcription factors eg C-MYC, C-jun, C-fos which then act to drive cell proliferation
MEK and ERK are both serine or threonine (not tyrosine) kinases

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16
Q

Turning off the MAPK pathway

A

Many ways to turn off MAPK pathway to stop cell growth:
-remove signal eg stop expressing EGF
-switch of receptor by Tyr phosphatase remove phosphorylation signal so receptors become inactive
-GTPase activating proteins drive inactivation of RAS and therefore stop signal
-dephosphorylation of targets by ser/thr phosphatases
Ultimate effect cell growth switched off
In tumours this is often deregulated eg RAS mutation

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17
Q

MAPK mutations and cancer

A

RAS is mutated 90% of prostate cancer
Commonly mutated in ovarian cancer too
RAS mutations in cancer always involve point mutations at specific sites (codons 12,13,21)
-eg G/T transversion (gly->val), typical carcinogen induced mutations in smokers or workers with occupational carcinogen exposure
RAF mutations common in melanoma (80%), lung and colorectal cancers:
-basal level kinase activity elevated 2-12 fold
-V600D and E mutations (most common)- mutants stimulate proliferation independent of upstream signals because mutation elevates activity RAF to turn on downstream signal

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18
Q

Summary of the PI3 (phospatidylinositol) kinase pathway

A

This pathway can be turned on by lots of signals eg insulin
One of the key molecules that is involved in this pathway is a lipid called PI : PI(3,4)P3, PI(4,5)P2
PI binds to membrane and when you have activation of the receptor you recruit and then activate a kinase called PI3 kinase
PI3 kinase phosphorylates PI to give it PIP3
Once we have 3 phosphorylations we can recruit downstream proteins called PDK1 and AKT
AKT is activated via phosphorylation
AKT is then able to activate a number of targets with the ultimate effect of driving cell growth, proteins synthesis and suppression of apoptosis

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19
Q

PI (phosphatidylinositol) and PI3-kinase

A

PI (phosphatidylinositol) is a phospholipid found in eukaryotic cell membranes
PI is phosphorylated to form PI(4)P which is in turn phosphorylated to form PI(4,5)P2
PI3K is activated by binding to phosphorylated Tyr residues on RTKs
PI3-K catalyses phosphorylation from PI(4,5)P2 to PI(3,4,5)P3

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20
Q

PDK1, AKT and mTOR

A

PI(3,4,5)P3 acts as a docking site for 2 proteins, PDK1 and AKT
Upon binding PDK1 phosphorylates and activates AKT
mTOR (as part of mTORC2) also plays an important role in activating AKT
AKT activates mTOR (as part of mTORC1) to stimulate cell growth via protein production/preventing protein degradation

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21
Q

Turning off the PI3-kinase pathway

A

Normal cells would want to turn off PI3K pathway once they’ve synthesised the appropriate proteins or undergone cell growth
The way pathway is tuned off:
-remove signal
-switch off receptor by Tyr phosphatase
-dephosphorylate PI(3,4,5)P3 to PIP2, PTEN (lipid phosphatases)
-dephosphorylation of targets by ser/thr phosphatases

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22
Q

Targeted treatments of these pathways

A

“Stratified” or “personalised” medicine;
-a more effective way of treating cancer by grouping patients according to their genetic mutation(s) and then targeting the signalling pathway to which it contributes

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23
Q

Receptor tyrosine kinases and cancer

A

Amino acid changes or partial deletions:
-constitutive tyrosine kinase activity
-loss of “receptor domain”
-growth factor independent signalling
Gene amplification and overexpression:
-increased expression of normal receptors- allows more signal binding which results in response amplification
-overexpression of aberrant receptors
Increased signaling

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24
Q

Targeting RTKs

A

Target antibodies to receptor:
-inhibit ligand binding
-inhibit dimerisation
-result in degradation of receptor
-induce cell killing of tumour cells
Small molecule inhibitors of RTK:
-molecules need to get inside the cells todo this

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25
Q

HER2 and breast cancer

A

HER2 belongs to the epidermal growth factor receptor EGFR family of RTKs
Orphan receptor (no known ligand). Heterodimerises with other EGFR family members
Amplification found in 30% breast cancers
Overexpression/increased copy number:
-poorer prognosis
-constitutive signalling

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26
Q

Targeting RTKs with antibodies

A

Trastuzumab (herceptin): does not block dimerisation, blocks internalisation, partial block of downstream signalling, promotes antibody dependent cytotoxicity
Pertuzumab: blocks dimerisation and activation, effective in cancers where HER2 is activated but not overexpressed
Cetuxumab, panitumumab: blocks ligand (EGF) binding, steric hindrance, promotes antibody-dependent cytotoxicity

27
Q

HER2 targeted treatments

A

Trastuzumab: inhibits internalisation/cleavage of HER, Ab-dependent cell toxicity, indication( metastatic breast cancer, adjuvant early breast cancer, advanced gastric cancer)
Pertuzumab: inhibits ligand binding and dimerisation, indication (metastatic breast cancers, neoadjuvant breast cancer)
Lapatinib: reversible ATP competitor (small molecule), targets EGFR as well, indication (advanced breast cancer)
Trastuzumab-emtansine: same as Trastuzumab plus MT inhibition and cell lysis. Indication (advanced breast cancer)

28
Q

EGFR targeted treatments

A

Cetuximab: inhibits ligand dependent activation. Indication (colorectal cancer, colorectal cancer with WT KRAS, head and neck with radiotherapy)
Panitumumab: inhibits ligand dependent activations, colorectal cancer, colorectal cancer with WT KRAS
Patients with KRAS mutations do not respond to EGFR antibodies, KRAS mutations usually mutually exclusive with EGFR mutations
Erlotinib (gefitinib): reversible ATP competitor (small molecule), response defined by certain mutations . Indications (lung cancer, pancreatic cancer)

29
Q

Resistance to EGFR inhibitors

A

Eg amplification of EGFR in glioblastoma, breast and lung cancer
Resistance to erlotinib and gefitinib: Thr790Met mutation prevents binding of inhibitor

30
Q

BCR-ABL: a fusion kinase

A

BCR is a protein that regulates the activation of GTP in the cell, its a Tyr kinase
It works with ABL (cytoplasmic Tyr kinase promotes cell survival and cell growth upon activation ) which is usually inactive in the cell because it has a self inactivation domain which stops activation of the kinase
In certain tumours especially hematopoietic tumours you get a fusion between the gene that encodes BCR and gene that encodes ABL resulting in BCR-ABL fusion kinase
During the fusion parts of the 2 genes are broken so get missing parts of each: usually miss some GTPase activation function of BCR and lose inhibitor function ABL
So ABL and BCR Much more active
Then get another kinase domain from ABL and BCR cross talking which further activate one another becomes even more active

31
Q

BCR-ABL fusion

A

Coiled-coil increases tyrosine kinase activity
Fusion kinase activates ABL signalling pathways
Fusion inhibits the SH3 regulatory domain leading to constitutive kinase activity
BCR recruits ABL to cytoplasmic signalling pathways including RAS pathway

32
Q

The (9,22) translocation leads to expression of BCR-ABL in CML

A

Resultant chromosome is known as the Philadelphia chromosome t(9;22)-(q34;q11)
The main tumour we see as a result of BCR-ABL fusion in chronic myeloid leukaemia
The reasons why BCR-ABL inhibitors good target for CML is that its completely abnormal and exclusively expressed in tumour cells

33
Q

BCR-ABL inhibitors

A

Gleevec (imatinib mesylate; Novartis)
-specific inhibitor of the BCR-ABL tyrosine kinase
Found by random screening for inhibitors of other tyrosine kinases
Competitive inhibitor of ATP binding to active site inhibits tyrosine kinases activity
Has dramatic effects in patients 80% patients with CML: responses in 53/54 patients at upper doses, 29 cytological responses including 7 complete cytogenetic remissions, remissions durable (18 months) chronic phase
Some patients experience resistance to imatinib and relapse:
-development of point mutations within try kinase domain of BCR-ABL
-tendency to relapse with resistant BCR-ABL mutants if treated in acute phase (blast crisis)
-second generation Tyr kinase inhibitors have been developed

34
Q

What is an oncogene

A

A gene that has the potential to cause cancer when mutated or overexpressed
Several hundred oncogenes have been identified
Derived from a normal cellular gene termed proto oncogene
“Activated oncogene”
Variant with abnormal expression or activity that positively contributes to cancer formation

35
Q

Proto-oncogenes and oncogenes

A

Protooncogenes are normal cellular genes that control critical processes such as proliferation, survival and differentiation
Typically function as growth factors, receptors, signal transduction molecules and transcription factors
Activated oncogenes over express these normal proteins or produce altered proteins with altered (usually constitutive) activity that contribute to the hallmarks of cancer

36
Q

Key features of oncogenes

A

Usually constitutively active i.e gain of function mutation
A single altered copy (allele) is sufficient ie dominant effect
Gain of function drives cell transformation

37
Q

Mechanisms of oncogene activation

A

Point mutations-> protein with altered characteristics eg EGFR, RAS, BRAF
Amplification of a genomic DNA region that includes the proto oncogene-> overexpression of the gene and increased amounts of protein eg MYCN., EGFR
Chromosome translocation that brings a proto oncogene under the control of a different promoter-> inappropriate gene and protein expression eg c-myc, BCL-2
Chromosome translocation that joins the 2 genes together-> creates a chimeric fusion gene and protein with novel characteristics eg BCR, ABL

38
Q

Oncogenic single nucleotide variants (SNVs) seen in cancer

A

EGFR- L858R (leucine 858 to arginine)
KRAS- G12C (glycine 12 to cysteine)
BRAF- V600E(valine 600 to glutamic acid)
PIK3CA- H1047R (histidine 1047 to arginine)
All lead to altered protein activity

39
Q

Receptor tyrosine kinases

A

58 human receptor tyrosine kinases RTKs
Examples include EGFR (ErbB1, Her1), ErbB2 (her2), VEGFR, ROS, RET
Control cell growth, survival, motility, differentiation, metabolism
Share a similar protein structure of an extracellular ligand binding domain a transmembrane helix and an intracellular tyrosine kinase TK domain
Dysregulation leads to many human diseases, especially cancer

40
Q

Common EGFR mutations

A

In Frame deletion eg del 747-752, increases dimerisation
Missense mutation eg L858R (leu>arg), increases kinase activity 50fold
When the receptor has one of these 2 mutations it becomes constitutively active and signals through downstream pathway constantly

41
Q

RAS is frequently mutated in cancers

A

RAS a molecular switch involved in signal transduction
Different RAS genes are associated with different cancers
Different cancers show variable patterns of KRAS, NRAS and HRAS mutations

42
Q

Oncogenic RAS mutations invariably involve Missense mutations affecting one of 3 codons (G12, G13, or Q61)

A

Mutations occur at just 3 amino acids in the entire protein sequence glycine 12, 13 and glutamine 61
This is because if we look at the 3D structure of RAS G12, G13, Q61 are all part of the same pocket in ED structure the GTP binding site

43
Q

How do these mutations affect RAS

A

RAS is involved in a signalling cascade from the RTK which ultimately results in changes in gene expression
RAS is switched between an on/off conformation depending on whether it is bound to GDP or GTP
-when it is bound to GTP, RAS active
-when its bound to GDP inactive
Mutations at G12, G13, Q61 at GTP binding site decrease GTP hydrolysis and so lock RAS in active GTP bound state
This leads to constitutive activation of RAS/MAPK pathway

44
Q

Mutations in RAS and EGFR are typical of those seen in many oncogenes

A

Mutations are clustered in specific regions of the gene (hotspots)
These regions encode important functional domains in the proteins
Most mutations are Missense mutations (amino acid substitution) resulting in a gain of function

45
Q

Oncogene amplification in cancer

A

MYCN- neuroblastoma -20%
C-MYC- small cell lung cancer- 15-20%, breast cancer 20%, ovarian cancer 20-30%, oesophageal cancer- 38%
Cyclin D1- breast cancer -20%, oesophageal cancer 25%
EGFR- glioblastoma -40%
All lead to protein overexpression from the amplified gene

46
Q

MYCN amplification in neuroblastoma

A

MYCN amplification detected by FISH

47
Q

N-Myc protein expression is increased in neuroblastomas with MYCN gene amplification

A

If you get a tumour sample and take to path lab they look for expression of the MYCN protein using immunohistochemistry
Lots n-myc in MYCN amplification

48
Q

MYCN amplification as a prognostic factor

A

MYCN amplification frequently associated with aggressive diseases, metastatic potential, therapeutic resistance and poor patient outcomes

49
Q

ERBB2/HER2 amplification in breast cancer

A

Associated with poor prognosis and reduced survival.

50
Q

Translocations leading to changes in oncogene expression

A

T(8;14) C-myc. Burkitt lymphoma
T(14;18) BCL2 follicular lymphoma
T(3;14) BCL6. Diffuse large B cell lymphoma
T(1;14) TAL1 T cell acute lymphoblastic leukaemia

51
Q

Burkitt lymphoma BL

A

Highly aggressive b cell lymphoma derived from mature B cells
Frequently presents as a fascial tumour
One of fastest growing tumours

52
Q

Translocations involving c-MYC are a hallmark of Burkitt lymphoma

A

All BL tumour cells carry a chromosomal translocation involving c-MYC gene on chromosome 8 and one of the immunoglobulin gene loci
T(8;14) MYC-IgH 85%
T(2;8) MYC-IgK
T(8;22) MYC-IgL

53
Q

Identification of t(8;14)(q24;q32)

A

How can we detect translocation
Using karyotyping-chromosomes stained with a dye and matched up based on size and banding pattern
Chromosome 8- where MYCN gene normally located
Chromosome 14- where normal immunoglobulin heavy chain is located
Theres a bit missing at end of C8 and extra on C14. C8-C14 translocation and as result place MYC gene next to immunoglobulin gene on this chomosome

54
Q

T(8;14) translocation places IgH enhancer adjacent to c-MYC

A

When you translocate onto C14 next to IgH enhancer you get overexpression of MYC
Because IgH enhancer very powerful in B cells as they need to produce lots of antibodies
So when you put MYC gene next to enhancer and promoter you end up with super high level of expression of MYC
Ki67 marker of cell proliferation

55
Q

C-MYC is a transcription factor that regulates >600 cellular genes

A

Genes required for:
-cell growth
-proliferation
-ribosomal synthesis
-protein synthesis
-metabolism
-energy generation
These are all hallmark of cancer
So if you deregulate MYC because it controls many of the proteins involved in different features of cancer the cell becomes cancerous

56
Q

C-MYC functions in cancer cells

A

Cell growth
Cell cycel
Apoptosis
Energy production
Anabolic metabolism
DNA replication
RNA biology and accumulation

57
Q

Translocations leading to fusion genes

A

T(9;22) BCR, cABL chronic myeloid leukaemia
T(8;21) RUNX, ETO acute myeloid leukaemia
Inv(2) EML4, ALK non small cell lung cancer
T(5;1) SQSTM1, NTRK1 non small cell lung cancer and many others

58
Q

BCR-ABL is a hallmark of CML

A

All CML cells have this translocation in it
Translocation between C9 and C22 -Philadelphia chromosome t(9;22)-(q34;q11)
Novel BCR-ABL fusion protein

59
Q

Proto-oncogene to oncogene

A

4 main mechanisms
Mutation in coding sequence- hyperactive protein made in normal amounts
Gene amplification- normal protein greatly overproduced
Chromosome rearrangement- nearby regulatory DAN sequence causes normal protein to be overproduced, fusion to actively transcribed gene greatly overproduces fusion protein; or fusion protein is hyperactive

60
Q

Why is this important

A

Genomic analysis of cancer cells:
Better understanding of the function of oncogenes
Detailed information about mutational landscape of cancers
Enables improved cancer diagnostic tests
Better prognostic information
Important for designing new targeted cancer treatments

61
Q

Era of precision cancer therapy

A

Move away from ‘one size fits all’ approach
Develop inhibitors of key oncogenes that drive tumour cell growth
Improve patient outcomes and reduce side effects
Examples include: BCR-ABL- imatanib, EGFR- gefetinib, afatanib, osimertinib, HER2- Trastuzumab, RAS- sotorasib
Ib-small molecule inhibitor.
Ab-monoclonal antibody

62
Q

BCR-ABL: a paradigm for targeted therapy

A

Imatinib (gleevec) is a specific inhibitor of the BCR- ABL tyrosine kinase
Competitive inhibitor of ATP binding to active site, inhibiting tyrosine kinase activity
Treated CML patients show 83% 10 year survival

63
Q

EGFR targeted therapies

A

First generation TKI eg gefitinib, erlotinib
Competitive ATP mimics, reversible binding, moderatively increase life expectancy
Second generation TKI eg afatinib, dacomitinib
Irreversible binding in the ATP pocket, binds to mutated version which causes resistance
Third generation TKI: osimertinib, binds more avidly to EGFR T790M mutants than wild type EGFR

CAN (11 decks)

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