Cancer Flashcards
1
Q
What are 4 defining features of cancer?
A
1) Cells have escaped normal limitations of external cue driven cell division
2) Can modify local environment to exceed natural defined tissue borders
3) Forms multicellular mass driven by “transformed” cancer cell
4) Have mechanisms to survive immune surveillance & cell death
2
Q
What can act as stimulants/limiters for cell growth?
A
Stimulunats:
Growth factors (EGF, FGF) & hormones (oestrogen, testosterone) activate signalling pathways.
ECM has collagen + laminin which regulate growth via chemical + physical signals
Limiters:
Chemical microenvironment is the cells niche (O2, temp, pH + chemical signals not being optimal can limit growth)
3
Q
Desrcibe a situational regulator of cell growth
A
Cytokines - small signalling proteins released by immune cells
Interleukins + interferons can promote/suppress division
e.g. IL1B promotes angiogenesis, decreasing proliferation
activating myeloid cells to produce pro-angiogenic factors like VEGF, and it can also directly stimulate endothelial cells to produce VEGF (via STAT/Nf-KB pathway)
4
Q
How can a cells local environment be modified so it exceed defined natural tissues borders?
A
ECM can be:
- degraded (matrix metalloproteinases)
- remodelled
- crosslinked (stiffer + stronger)
ECM receptors expressed in cancer cells.
Angiogenesis promotes blood vessel formation
5
Q
How can a ‘transformed’ cancer cell form a multicellular mass?
A
Stepwise/transformative model - multiple mutations accumulate.
Mutation 1 - remove negative regulator of cell cycle
Mutation 2 - activates +ve regulator of cell cycle
Mutation 3 - inhibits cell death
-> additive effects cumulative mutations
6
Q
How do cancers escape the immune system?
A
Immune depeletion/modification creates imbalanance which causes proliferative cell growth.
Elimination -> equilibrium -> escape
Greater the heterogeneity/genetic instability the more like to escape (immune selection)
7
Q
What mechanisms do cancers employ to survive immune surveillance & death?
A
- Mutations in genes that regulate apoptosis e.g. p53
- Upregulation of anti-apoptotic proteins e.g. BCL-2, MCL-1 which inhibit initiation of apoptosis
- Downregulation of pro-apoptotic proteins (BAX, BAK)
- Activation of survival signalling pathways e.g. PI3K/AKT/mTOR or RAS/RAF/MEK/ERK
- Altered balance of death receptors & ligands e.g. FAS, TNFR1
8
Q
Cancer in epithelial cells
A
Line surface of internal organs & glands forming barriers.
-> wide variety of carcinomas inc. lung, colon + breast
9
Q
Cancer is mesenchymal cells
A
Form connective tissue between many cell types e.g. bone, muscle, fat
-> sarcomas e.g. osteosarcoma (bone) & leiomyosarcoma (smooth muscle)
10
Q
Cancer in hematopoietic cells
A
Give rise to blood cells + are found in bone marrow
-> cause leukemias e.g. acute lymphoblastic (ALL), or chronic myeloid (CML)
11
Q
Cancer in lymphoid cells
A
Type of WBCs are important for immune system.
-> causes lymphomas e.g. Hodgkin & non-Hodgkin
Cancers acan also arise from germ cells, gliomas, meningiomas, pituitary tumours, neural blastomas + schwannomas
^always have an aspect of their origin (specificity in natural programming)
12
Q
What is the clinical presentation in cancer diagnosis?
A
Observation of symptoms and signs suggestive of cancer.
13
Q
What is a biopsy?
A
Removal of tissue for examination under a microscope to confirm the presence of cancer cells.
14
Q
What are imaging studies used for in cancer diagnosis?
A
To visualize the location and extent of the cancer using X-rays, MRI, CT Scans, PET Scans, and other imaging tests.
15
Q
What do laboratory tests in cancer diagnosis detect?
A
Elevated levels of cancer markers or the presence of abnormal cells in the blood.
16
Q
What is staging in cancer diagnosis?
A
Determination of the size and spread of the cancer to help guide treatment options.
17
Q
What does prognosis refer to in cancer diagnosis?
A
Prediction of the likely outcome of the disease based on factors such as stage, location, type of cancer, and overall health.
18
Q
Why is early diagnosis key in cancer treatment?
A
It allows for earlier surgery and successfully mitigates the spread or removes cancer.
19
Q
What does the term ‘hypoxia’ refer to in the context of cancer cells?
A
A condition where cancer cells are deprived of adequate oxygen.
20
Q
Fill in the blank: Cancer cells shunt energy production to _______ only, using lots of glucose.
A
[glycolysis]
Away from krebs/citric acid cycle. Less ATP produced compared to normal cells w/ lots of O2
21
Q
What is the role of the GLUT1 transporter in cancer cells?
A
To concentrate large amounts of glucose due to hyperactivity in cancerous cells.
22
Q
True or False: Men are 1.5 times more likely to get cancer than women.
A
True
23
Q
What are the causes of difference in cancer susceptibility between men & women?
A
- riskier behaviours (smoking, alcohol, no activity)
- different biological susceptibilities (anatomy)
- delayed diagnosis & treatment (less likely to seek medical attention)
- occupational exposure
- genetic susceptibility
Multivariant causes
24
Q
What can cause false diagnoses of cancer?
A
Thyroid and prostate conditions can be wrongly diagnosed as cancerous.
25
What is a fluorescent glucose analogue (FGD-PET) used for?
To visualize tumors in the body that concentrate large amounts of glucose, due to GLUT1 hyperactivity
26
Role of Ras signalling & hypoxia in cancer development?
Hyperactivate genes/proteins in cancerous cells
27
Which therapies do females respon better to than men?
Many:
- surgery
- radiotherapy
- chemotherapies
- EGFRi targeted therapy
BUT some immunotherpies see better response in men
28
Define epidemiology in cancer
Distirbution & determinants in cancer patients
29
Why does cancer incidence increase w/ age?
Accumulative exposure (migration data osaka -> hawaii shows cancer mainly exposure driven), behaviours & genetic mutations
## Footnote
42% cancers preventable
30
What types of cancer are largely associated with alcohol consumption?
Epithelial tissues in mouth, throat, oesophagus, breast, liver, colon, and rectum
## Footnote
These cancers are influenced by the toxic by-products of alcohol metabolism.
31
What toxic by-product is created when the body breaks down alcohol?
Acetaldehyde
## Footnote
Acetaldehyde can damage DNA and proteins in cells, increasing cancer risk when it accumulates.
32
How does alcohol consumption affect hormone levels?
It can increase hormone levels, such as oestrogen
## Footnote
Elevated oestrogen levels can stimulate the growth of certain cancers, particularly breast cancer.
33
Why is alcohol considered lipid soluble?
Because it can be stored in fatty tissues
## Footnote
This property affects how alcohol impacts the body and its tissues.
34
List three effects of alcohol on the body.
* Inflammation
* Oxidative stress
* Weakened immune system
## Footnote
These effects can contribute to cancer development.
35
What enzyme converts ethanol into acetaldehyde?
Alcohol dehydrogenase (ADH)
## Footnote
This enzyme plays a key role in alcohol metabolism.
36
What is the role of ALDH in alcohol metabolism and how genetic variations affect cancer risk?
ALDH converts acetaldehyde into acetate - helping reduce toxicity of alcohol metabolism
People with mistakes in the gene coding for ALDH may accumulate acetaldehyde
37
What is a major way smoking contributes to cancer?
By introducing harmful chemicals that damage DNA or make it harder for cells to repair DNA damage
## Footnote
Smoking primarily affects lung tissue but is linked to various other cancers.
38
What is meant by 'reversible, accumulated damage' in the context of alcohol and cancer?
Damage can be reversed to some extent but accumulates with higher concentrations over time.
It is concentration dependent
## Footnote
This indicates that while some effects can be mitigated, ongoing exposure increases risk.
39
Describe the association of high BMI to cancers
Correlated to some cancers e.g. lung & malignant melanoma, but not others.
- chronic inflammation due to fat cell removal
- insulin resistance
- hormonal alterations
- organ pressure: structural changes -> waste products build up
- poor nutrition: less antioxidants + more free radicals
40
How can obesity cause cancer?
## Footnote
Not proven to cause, just correlates with some
Fat cells increase inflam & make extra hormones/growth factors
-> cells in body divide more often
Increases occurnece of cancer cells which can continue to divide + cause tumour.
## Footnote
ALL causes relatively indirect, no direct link to DNA alterations
41
What is a direct cause of cancer?
Something that causes direct mutation to DNA, altering expression.
Somatic mutations - in developed tissues, carrier has the change
Germline mutations - in ovaries/testes, produce offspring carrying the change
## Footnote
Somatic mutation accumulation is stepwise model, leads to predictable unpredictability (many different outcomes)
42
Monoclonal vs polyclonal tumour
Monoclonal - originates from a single mutated cell, all cancer cells within the tumor are genetically identical + descended from that one cell.
Polyclonal - arises from multiple mutated cells, resulting in a tumor with genetically diverse cancer cell populations originating from different progenitor cells
## Footnote
1 cell lines vs many cell lines
43
What is the primary cause of treatment failure?
Heterogeneity - more exposure, more changes, more variants.
e.g. bipsy of polyclonal tumour will skew identification, reality will be many tissues types
44
What are some carcinogens?
Carbon based/organic carcinogenic agents, integrate into metabolic pathways + causes failures
e.g. smoke, grilled meats, tobacco, paints & dyes (benzene ring structures)
## Footnote
Some only actve after processing in liver
45
Ames test
For suspected carcinogens. Quick, simple + inexpensive to scren large no. of chemicals for mutagenicity.
When a gene in Salmonella is knocked out, hystidine synthesis prevented:
- this leads to a single point nonsense or frameshift mutation that converts his+ to his-.
If chemical mutagenic, will convert his- back to his+
## Footnote
Only indicative so followed by in vivo testing in mice.
46
Pros of Ames test
* Quick and inexpensive
* High-throughput
* Uses well-established model organism: Salmonella typhimurium,
* Simple to perform
* Reliable
* Non-animal
47
Limitations of Ames test
* Limited predictive power: indicate whether a chemical is mutagenic, it cannot predict toxicity or the specific types of mutations that may occur in humans.
* May not detect all mutagens: The test only screens for mutagens that cause frameshift mutations.
* May not detect all mutagens: Some chemicals that are mutagenic in higher organisms may not cause mutations in the bacteria
* May give false positives: Some chemicals that are not mutagenic in higher organisms may cause mutations in the bacteria
* May not detect all mutagens: anything toxic and kills bacteria is not assessed for mutagenicity
48
What is a proto-oncogene?
The normal cellular version of an oncogene
## Footnote
Proto-oncogenes regulate cell growth and division.
49
What is the role of complementary viral DNA in relation to proto-oncogenes?
Used to hijack proliferation control for greater viral replication.
It targets src (non-RTK) gene, nicks it.
50
What happens when viruses capture proto-oncogenes?
'Capture' - accidental integration next to proto-oncogene (c-src)
-> can drive uncontrolled cell proliferation
## Footnote
This can lead to cancerous growth.
51
What is the function of oncogenes?
Act as accelerators of cell proliferation - increase it
## Footnote
They can arise from gain-of-function mutations in proto-oncogenes.
52
What is the significance of the C-Src proto-oncogene?
It can be removed by the virus & damaged during exit
## Footnote
This can lead to the generation of a fused ALV-Src RNA transcript.
53
What is the Bcr-Abl fusion protein?
Formed by reciprocal translocation between chromosomes 9 & 22.
-> located within philadelphia chromosome (an even smaller Chr 22)
## Footnote
This fusion is implicated in chronic myeloid leukemia.
54
What are some types of oncoproteins involved in cellular signaling?
* Non-receptor TK
* TFs
* EGF RTK
* Small G protein
## Footnote
Oncoproteins can have various roles in signaling pathways.
55
What is the role of the Abl protein?
A non-receptor tyrosine kinase that induces allosteric change
## Footnote
It adds negative charges to tyrosine residues, activating downstream signalling proteins/enzymes.
56
What happens when the N terminal sequences of Abl are missing?
The fusion protein becomes an active tyrosine kinase
## Footnote
This can lead to enhanced signaling and proliferation.
57
Fill in the blank: Oncogenes suppress _______.
apoptosis
## Footnote
This contributes to the survival of cancer cells.
58
True or False: C-Src is the viral version of the cellular version Src.
False
## Footnote
C-Src is the cellular version, while V-Src is the viral version.
59
What cellular pathway does Abl ordinarily activate?
Ras signaling pathway (e.g., MAPK)
## Footnote
This pathway is crucial for cell proliferation - constitutively active when oncogenic. e.g. Bcr-Abl, v-Abl
60
What are the roles of SH2 and SH3 in Abl?
SH2 binds Tyr residues (allows it to bind other proteins or induce conformational changes)
SH3 interacts w/ Pro residues -> adhesive binding domain
61
What causes Abl to be oncogenic?
- fusion & myristoylation of Gag -> targets v-Abl to mem where it is hyperactivated
- loss of SH3 domain in v-Abl (reduced size in Bcr-Abl)
-> contitutively active, no signal needed
62
Describe the structure of inactive Abl.
Contains N-lobe, C-helix, and C-lobe.
SH3 clamped to C-lobe
63
What is required for Abl to be fully active?
Phosphorylation of y245 and y412.
-> disengages SH3 from Pro residues, conformational change activates protein.
64
What is the significance of Bcr-Abl as a fusion protein?
Fusion protein removes clamp mechanism (encoded at start of SH3 domain)
- contituitve active form of Abl
65
What are potential proto-oncogenes involved in?
Growth regulatory pathways.
66
What types of proteins are associated w/ cancer?
Those involved in cell proliferation, short-circuit when mutated so constitutively active.
- TFs
- Kinases
- Signalling mols
- Cell death regulators
67
Possible cancerous effect of regulatory & non-coding regions being mutates
Promoter GoF can lead to overexpression of proto-oncogen product (oncogenic)
e.g. myc translocation next to very active promoter
68
Role of Bcr-Abl in proliferation signalling
Phosphorylates Tyr residues on target proteins (CRKL, Gab2, SHP-2, and β-catenin) using ATP - inducing cell proliferation causing CML
Gleevec mimics ATP binding to site on Bcr-Abl (competitive antagonist), prevents phosphorylation.
69
Tumour suppressor disocvery
Henry Harris - fusion of normal cells w/ cancer cells suppresses the cancer
Tumour suppressors negatively regulate cell cycle, if lost then cells free to proliferate.
Also act as 'caretakers' of genome - DNA repair after mutation, if lost then allows accumulation of mutations in DNA.
70
Retinoblastoma
Malignant tumour of developing retina.
Inherited null Rb mutant -> gene dosage effect (happloinsufficiency)
Loss of 1st allele causes destabilisation but loss of 2nd causes retinoblastoma (2 hit hypothesis)
71
Is Rb a tumour suppressor?
Yes, transcriptional repressor, but additional mutation required - co-operativity.
- Regulates cell cycle, G0 - G1, holds back cell cycle by binding E2F blocking G1/S transition when dephosphorylated
- Recruits HDACs locking genes in repressive state (heterochromatin)
- When phos by CDKs, E2F free to activate as no Rb bound
72
What is the primary function of the p53 tumour suppressor?
Suppresses G1-S phase transition and arrests cell cycle in G1 upon DNA damage.
Induces p21 (cyclin dependent kinase inhibitor) - switches on repair genes or induces apoptosis
## Footnote
p53 is often referred to as the 'guardian of the genome' due to its critical role in maintaining genomic stability.
73
In what percentage of cancers is p53 mutated?
Mutated in 1/2 all cancers.
## Footnote
This high mutation rate highlights the importance of p53 in cancer biology.
74
What happens when p53 is deleted?
Leads to cell survival and loss of G1 arrest.
## Footnote
The deletion of p53 removes the regulatory control it exerts on the cell cycle.
75
What triggers the accumulation of p53 protein?
DNA damage, e.g., UV damage.
## Footnote
Normally, p53 is rapidly degraded, but its levels increase significantly in response to DNA damage.
76
What role does p53 play in the cell in response to damage?
Induces repair genes or apoptosis.
## Footnote
p53 can initiate cell repair processes or lead to programmed cell death if damage is irreparable.
77
What is the role of co-activators in p53 function?
Makes genes more accessible by recruiting DNA remodeling proteins.
## Footnote
This accessibility is crucial for the transcription of genes involved in cell cycle regulation and DNA repair.
78
What does p53 activate upon binding to the p53 response element?
Three pathways: death, repair, growth arrest.
## Footnote
These pathways are essential for maintaining cellular health in response to stress.
79
What is the structure of a receptor tyrosine kinase (RTK)?
Extracellular region, transmembrane region, intracellular tyrosine kinase domain.
## Footnote
This structure allows RTKs to transmit signals from outside the cell to the interior.
80
Which RTK is overexpressed in breast cancer?
HER2.
## Footnote
HER2 overexpression is associated with aggressive forms of breast cancer.
81
What induces a conformational change in RTKs?
Binding of peptide ligands.
## Footnote
This conformational change activates the tyrosine kinase domain, leading to downstream signaling.
82
Fill in the blank: p53 normally undergoes rapid _______ but accumulates in response to DNA damage.
degradation.
## Footnote
The rapid degradation of p53 is a key regulatory mechanism that is disrupted when DNA damage occurs.
83
What is the primary role of the intracellular tyrosine kinase domain in RTKs?
Phosphorylates proteins.
## Footnote
This phosphorylation is essential for propagating the signal initiated by ligand binding.
84
What does RTK actvation lead to?
Activation of RAS-MAPK pathways:
- ligand binding induces phos of SH2 domain on GRB2
- recruitment of SOS complex to GRB2 via SH3 domain
- activates RAS which activates downstream cell proliferation pathways
## Footnote
SOS catalyses GDP-GTP exchange on GTPase RAS
85
Is HER2 a tumour suppressor?
No it is an oncogene, must remain in inactive state for constitutive cell proliferation.
## Footnote
Patients who are HER2 +ve are generally oestrogen receptor -ve so cannot use tamoxifen to inhibit.
86
How can we block HER2 activity in breast cancer?
Monoclonal antibodies
Pertuzumab - inhibits dimerization of receptor via binding the dimerisation domain
Trastuzumab - inhibits signal transduction, long lasting immune reactive response -> reduces circulating Tregs
## Footnote
Inhibit ability of RTK to be active
87
Role of co-operativity in cancer development
2 hit hypothesis
oncogenes + tumour suppressors drive transformation of a cell to become cancerous
88
How can a multicellular mass be driven by a transformed cancer cell?
Mutation 1 - removes negative cell cycle regulator (tumour suppressor)
Mutation 2 - activates positive cell cycle regulator
Mutation 3 - inhibits cell death
+ additive effects of TME
## Footnote
Stepwise model due to mutation accumulation -> explains increase in cancer incidence w/ age
89
Cancer hallmarks
- sustain proliferative signalling
- evade growth suppressors
- metastasis + invasion
- replicative immortality
- angiogenesis induction
- resisting cell death
## Footnote
So single oncogene needs to initiate many processes
90
What are the pressure points in cell signalling?
All pathways via similar set of kinases & transcription factors
e.g. SRC, PI3K, RAS, JAK/STAT
Vs many receptors/ligands
e.g. EGFR can phosphorylate whole array of second messengers - signalling hub, can engage many different signalling mols, activate multiple pathways.
e.g. GRB2, SRC, PLC, SHC
91
What is MYC?
Oncogenic TF
Basic helix-loop-helix TF
-> cell proliferation, differentiation, apoptosis, and metabolism
## Footnote
Constitutively & aberrantly expressed in >70% human cancers.
92
Molecular mechanism of MYC
It forms a heterodimer w/ Max protein, this complex binds to specific DNA sequences, enhancer boxes (palindromic - CACGTG)
Half-life of only around 20 minutes + rapidly degraded by the proteasome (very potent -> fine tuning regulation)
93
What is FISH used for?
Fluorescence In Situ Hybridization - test for gene amplification in potentially cancerous cells -> biomarker detection
94
How is MYC stabilised for consititutive activation?
Phosphorylation at Serine 62 (S62) generally leads to MYC stabilization
Deubiquitination enzymes like USP28 and USP36 can further stabilize MYC by counteracting the U3 ubiquitin ligase Fbw7
-> MYC inhibits its own degradation
## Footnote
PI3K/AKT & RAS/RAF signalling can comstituively activate MYC
95
What cancers is MYC amplification most prominent in?
Breast - 15%
Ovarian - 33.2%
96
Deregulated vs physiological MYC
Deregulated Myc binds lower affinity non-target genes + E-box -> more signalling pathways activated.
-> tumorigenesis
## Footnote
Dysregulated chorus of signalling
97
Describe different Myc target genes + their functional groupings
Cell adhesion/cytoskeleton - a3B1, integrin, cdc42
Transcription - E2F
Translation - eIF-2a
Signal transduction - Wnt pathway
miRNAs - Let7 (tumour suppressor, negatively regulates RAS in C. elegans)
98
Give a few examples to decrease Myc activity
- Transcription e.g. JQ1
- Signalling to MYC e.g. via mTOR, rapamycin inhibits
- Stabilisation e.g. MLN8237 inhibits AURKA -> destabilisation
- Binding to Max protein e.g. Omomyc
99
Do Myc amplification & mutant RAS co-operate?
Yes - lymphoma mouse model -> sharp decrease in survival when cooperation between oncogenes
- situationally dependent tranformation of cancer cells
Vs lung cancer mouse model:
No additive effect of both oncogenes - similar durvival to only KRAS mutant
100
What is oncogene co-operation?
Different oncogenic drivers may cooperate to promote disease progression by acting on the same oncogenic pathways or by the convergent effects on independent pathways perturbed by individual mutations.
101
Autocrine driven co-operation/ signal diversification
## Footnote
Oncogene co-operation
Mutant Ras induces TGF-a synthesis -> ligand that is secreted + binds EGFR -> activation of multiple pathways
Cancer cells want to activate different branches if signalling pathways (2 main branches):
- Common to find mutations in BRAF & PI3K/PTEN loss -> causes dysregulated proliferation survival in cancer
- Occurs frequently when targeted inhibitors used to block other branch over the other
102
RTK swapping
## Footnote
Oncogenic co-operation
Mutant RTKs synergise to activate similar pathways - commonly occurs when one RTK inhibited
EGF (cetuximab, erlotinib) & VGF (pazopanib, sunitinib) inhibition
Activates alternative signalling pathway Pi3k/AKT
## Footnote
Ligand production feeds back on itself through same pathways & provides opportunity to alter tumour microenvironment - TME manipulation via immune evasion.
103
How does co-operativity induce heterogeneity?
Each oncogenic driver may be signalling through different co-operative pathways - autocrine & paracrine signalling.
SO heterogeneity in polyclonal tumours also provides co-operation between oncogenic drivers - significant cross talk.
104
What is gene expression profiling
Technique used to query expression of thousands of genes simultaneously, used to accurately classify tumours.
- Info derived is often predictive of patients clinical outcome + treatment pathway
105
Gene expression profiling for breast cancer
HR+/HER2+ are present 68% breast cancer patients
HR-/HER2+ 10%, HR+/HER2- 10%
106
What is HR+?
## Footnote
Breast cancer
HR+ means tumour cells have receptors for oestrogen or progesterone -> promote growth of HR+ tumours
107
What is HER2+?
HER2 (human epidermal growth factor receptor 2), HER2+ means tumour cells make lots of HER2+
HER2 is RTK which plays role in cell growth, differentiation + survival. Overexpression/ amplification can lead to growth/division.
108
WHat are typical treatments for HER2+ cancers?
Targeted therapies e.g. trastuzumab (Herceptin) & pertuzumab (Perjeta)
109
Immunohistochemistry for classification of breats cancer types
For ER, HER2 & KI-67.
-> subjective profiling
ER+/HER2- -> Luminal A & B, treated w/ tamoxifen hormonal kit, imatinib therapy
ER-/HER2- -> basal like, can use Iaptanib + PARP inhibitorys
Er-/HER2+ -> Her2+, can use herceptin as treatment
## Footnote
KI-67: proliferation marker which can indicate aggression, useful for determining treatments
110
What does future of gene expression profiling look like?
Can look at known biomarkers for cell behaviour - oncotype DX (21-gene assay)
- Designed to quantify risk of recurrence
- 16 genes (+5 reference genes) were selected that correlated with proliferation and endocrine response
If recurrence score high then get more treatment (chemo)
111
Functional vs cancerous tissue differentiation properties
Cancers generally move from differentiated states towards proliferative/undifferentiated phenotype.
Normal tissues maintain functionality w/ continual replacement of dfferentiated cells.
## Footnote
Despite no strict dichotomy, proliferation & differentiation are at different ends of the spectrum.
112
List 6 advantages of cancer cells being undifferentiated
* Proliferation: Undifferentiated cells have high capacity for self-renewal/can divide rapidly.
* Resistance to apoptosis
* Invasion and metastasis: can invade surrounding tissues and migrate to other parts of the body -> have retained some properties of embryonic cells, which can migrate during development.
* Increased plasticity: more adaptable -> can respond to changes in their environment more easily.
* Decreased immune surveillance: reduced expression of surface markers that would normally make them recognizable to the immune system as abnormal/foreign.
* Metabolic plasticity: altered metabolism so can adapt to different nutrient and oxygen conditions.
113
What is a key factor for cell cycle control?
Signalling integration
Collection of differentioation, growth, motility & survival factors + nutrients
-> controls DNA replication/cell division OR differentiation, survival + migration
114
What 2 different ultimate cell fates?
Enter G0 (quiescent state)
Enter active cell cycle -> programming of cell cycle phase
115
2 properties of the cell cycle
It is committed & unidirectional
-> checkpoints dtermine progression
## Footnote
Regulated by tight program of protein complex expression - cyclins
116
True or false: CDK concentration fluctuates furing cell cycle
FALSE - CDKs relatively constant in their expression, cyclins expression changes + activate CDKs via complex formation
117
Role of different cyclins
D: binds CDK4/6 driving early G1 up to restriction point
E: binds CDK2 late G1 after R point for S-transition
A: binds CDK2 during early S phase then CDK1 for G2 transition
B: binds CDK1 in G2 -> mitosis
## Footnote
CDC2 = CDK1
118
Describe unique features about the cell cycle phases?
G1 - only period where cells responsive to mitogenic growth factors
S - largest metabolic requirement to duplicate genome
G2 - double number of chromosomes for too long -> genome toxicity, too many deleterious genes
119
What controls transcriptional of cyclin D1?
Mitogenic signalling from multiple sources:
- RTK signalling via Grb2/Sos/Ras
- Wnts & Hedgehog signalling
- NF-kB activation
- cytokine activating JAK/STAT
- Growth factors -> Sp1
Promoter of cyclin D1 is vital for cell cycle progression.
Multiple TFs activate cyclin D promoter from different receptors & ligands.
## Footnote
Cyclin D activation is pressure point of cell cycle.
120
Translational control of cyclin D
Can indirectly phosphorylate Ribosomal protein S6 -> 40S subunit formation
- inhibited by rapamycin
Activates eIF4E for translation
## Footnote
It is a mtebaolic sensor complex that monitors nutrient levels
121
How is cycling D stability controlled?
Mitogenic signalling
RAS/Pi3K, via Akt phos GSK-3 inhibiting addition of Thr-286-phosphate tag to cyclin D.
SO it cannot be translocated from nucleus -> cytoplasm for UPS degradation via ubiquitin E1 enzymes
## Footnote
UPS - ubiquitin proesome system
122
What are the 3 levels of control over cyclin D expression?
mRNA levels -> mitogen induced TFs + promoter binding
translation -> nutrint dependent via mTOR
Stability/degradation -> mitogenic signalling can inhibit UPS
123
What is the role of GSK-3β in cyclin D proteolysis?
GSK-3β, a serine/threonine protein kinase - phosphorylates it at Thr-286
-> trasnlocation from nucleus to cytoplasm
124
Role of Rb in repressing S phase
Binds E2F & DP transcription factors on E2F site
- passive repression of downstream target genes by sitting on promoter sites
- active repression by HDAC action
Represses S phase proteins inc cyclin E expression
125
How is Rb repression inhibited, activating S phase?
Active CDK4/6 phosphorylates Rb -> no longer represses genes required for S phase
Rb dissociates from E2F & DP -> activation of S-phase genes & cyclin E (binds CDK-2 which also inhibits Rb repression)
- Feedforward activation until S phase initiated
## Footnote
During S-phase, TFs are degraded + Rb remains hyperphosphorylated until end of mitosis (dephosphorylated)
126
What are the brakes of the cell cycle?
Absence of growth factor signalling or nutrients (no mTOR function) & CDKIs.
127
List some CDKIs
p15, 16, 18 & 19 inhibit D-CDK4/6
p121, 27 & 57 inhibit E, A & B kinase complexes
128
How are CDKIs induced and antagonised?
Induced by growth suppressors e.g. TGF-beta, p53 -> increase in p21
ANtagonised by oncogenes e.g. MYC & AKT, but they can also produce CDKIs
129
How can p27 be removed?
Phosphorylation of Thr-187 -> targets it for ubiquitylation by proteosome
- requires formation of a trimeric complex with the cyclin and Cdk subunits.
- cyclin E/Cdk2 or cyclin A/Cdk2 mediated
(Montagnoli, 1999) used specific T-187 p27 Ab to show -> it is cell cycle dependent
## Footnote
Switch from cyclin D -> cyclin E is ultimate
130
Does cyclin E expression affect survival?
Higher cyclin E expression in breast cancer patients reduces chance of survival - loss of cell proliferation controls.
Constant feedforward mechanism -> persistent S phase, genomic instability (chromosomal misalignment/mis-segregation, can form >2 cells in mitosis)
Increase heterogeneity -> diversity promotes adaptation to treatments/nutritional status/new environments.
- more flexible & resilient TME
131
The dysregulation of cell cycle related factors are common hallmars of many different cancers?
LoF - Rb, p16
GoF - cyc D1, CDK4/6
## Footnote
Also cyclin E & p27
132
What are the 4 main cell cycle checkpoints?
DNA damage:
- entrance to S phase blocked if genome damaged
- replication also blocked if genome damaged
Entrance to mitosis cblocked if DNA replication not completed.
Anaphase blocked if chromatids not properly assembled on spindle
133
What are the options when a checkpoint is reached?
- Continuation if damage/error is repaired
- Apoptosis, elimination of problem (accumulation of death signals)
- Senescence (viable, active)
134
Guve an example of signal integration in the cell cycle
TGF-beta is a natural growth suppressor - activates p15 transcription (inhibs D-CDK4/6) & weakly activates p21.
So competes with Pi3K/Akt signalling inhibition on p21
135
How is the R point (G1/S transition) ultimately controlled?
Balance of multiple stop and go signals
Ultimately all goes via pRb
136
What is the restriction point?
Threshold that determines cells commitment into the cell cycle + progression to G1- important as genome replication is energetically expensive (large ATP use).
Switch from CDK4/6 -> CDK2 (cyclin D -> E) determines if cells enter S phase or if they enter quiescence or senescence (cell remains + can age)
137
In what way does signalling determine phenotype?
Digital exression - all or nothing
138
RTK signalling
Initiates intracellular signalling pathways via ligand/receptor dimerisation.
- stochastic element of interaction
139
What are the 2 types of RTK transphosphorylation?
Phosphorylation of receptor: activation loop initially phos (conformation change juxtamembrane domain -> catalytic cleft accessible), loop can then phosphorylate multiple different sites on receptor
Cross phos of adaptor kinases: e.g. Tyk2 & Jak1 can phos each other & the receptor -> activating variety of functions
140
Overexpression vs Consititutive activation
Constitutive activation (always on, due to mutations affecting structure), does not need ligand e.g. activation loop
Overexpression -> hyperactivation (difficult to switch off), still requires ligands, increased probability
141
How can RTK signalling become oncogenic?
- Amplification can cause genomic overexpression (translocation of chromosome)
- Transcriptional upregulation via oncogenic signalling
- Loss of inhibitory domains
- Mutations in key residues
## Footnote
Non-small cell lung cancer (NSCLC) - mutations in EGFR approx 10-15%
Glioblastoma - mutations in RTK gene known as EGFRvIII are found ~50% cases
142
Paraccrine signalling in RTKs
Cell-cell communication acting locally on neighbouring cells - TME can increase RTK signalling.
It coordinates cellular responses within tissues by regulating cell proliferation, differentiation & survival.
e.g. NTs, growth factors & cytokines
EGFR in ep cells activated by TGF-a -> secreted PDGF, activates PDGFR on mesenchymal cells -> secrete TGF-a
143
Why does RTK signalling need to be tissue & ligand restricted?
Common dysfunctional phenotype of cancer cells is expressing receptors not associated w/ tissue of origin
e.g. PDGF can induce siganl responses in endothelial, CSMCs, fibroblasts, other mesenchymal cells, glial cells.
144
What method of RTK signalling is defective/cancer inducing?
Autocrine - Cancer cells express ligands for their own receptors. RTK signalling can drive autocrine growth factor production.
EGF activates Ras signalling -> TGF-a synthesis + autocrine secretion where it binds EGFR -> release of EGF
## Footnote
EGF-R & TGF-a almost always co-localised/expressed because Ras signalling will produce TGF-a (produce own ligand).
145
Give some examples of human autocrine growth factors
TGF-a binds EGFR -> squamous cell lung, breast, prostate, pancreatic, adenocarcinoma
IL-6 binds IL-6R -> myeloma & HNSCC
IL-8 binds IL-8R -> bladder cancer
## Footnote
Not just autocrine though, can signal to other cancer cells/clones in a polyclonal tumour - ligand production can signal to other receptors in a polyclonal tumour.
146
Domain homology across RTKs
All have intracellular tyrosine kinase domain (PDGF, VEGF & FGF have kinase insert region)
EGF & IGF have extracellular cysteine rich domains (IGF linked by disuplhide bridges)
NGF, PDGF, FGF & VEGF have extracellular immunoglobin (IgG) like domains -> interaction w/ immune system
## Footnote
Ech R has IgG like domain, cystein domain & dibronectin type II-like domain
147
Are tyrosine kinases highly conserved?
Yes - even w/ non-receptor TKs
Src is regulator of cellular morphology - adhesion + ECM, functions as RTK domain
148
Give an example of an RTK that needs a cofactor to actuvate
FGFR needs heparin as well as FGF1/2
149
How do gene fusions lead to constitutive RTK activation?
RTK Ros + Fig protein binding -> constant firing of RTK as fusion protein formed (strong affinity of Fig domains)
- Fig is regulatory protein , disrupts normal Ros function
Common fusion partner examples:
FGF-R-3 to IgH + Tel
PDGF-Ra to BCR
TrkA & Met receptors bind Tpr
## Footnote
fusion event, leads to constitutive activation
e.g. Bcr-Abl for CML, Eml4-Alk for lung adenocarcinomas (prominent in non-smokers)
150
How do adaptor proteins & domains allow specific docking during intracellular RTK signalling?
Docking proteins (SH2) have different domains that want to bind phosphorylated Tyr
2 pocket model -> SH2 has binding site for pY AND a binding site for flanking amino acid side chains
## Footnote
SH - Src homology
151
Roel of SH2 & SH3
SH3 binds proline-rich oligopeotides on PDGF-R (Src activator region) when activated - linker segement when not
SH2 binds phosphotryosines
## Footnote
Location of binding sites determines signalling pathways activated.
- Require SH2 if they bind directly to RTK
- Require SH3 if they bind each other or other kinases
e.g. Fps & Syk only have SH2, Src & Grb2 has both
152
Src activation during RTK signalling
Normally self-inhibited: SH2 bount pY on C -terminal Y527, SH3 bound to linker segment
Complex rearranged: Csk phosphatase dephosphorylates Y527 on Src tyrosine kinase, SH2 free to bind pY on Src activator, SH3 binds proline rich oligopeptide adjacent -> storng affinity binding
Y416 on activation loop is autophosphorylated -> conformational change revealing catalytic cleft between N & C lobe of kinase domain
## Footnote
FAK is another adaptor kinase
153
Signalling adaptors for Ras activation
Either Grb2 bound to SH2 OR Shc & Grb2 bound to distinct SH2 domains
-> SH3 activated to bind Sos (GEF) which promotes GTP exchange for RAS activation
154
Describe the GTP cycle (exchange) for Ras activation
Inactive Ras bound to GDP.
- GEF (Sos) stirggers stimulatory signal -> GTP exchanged for GDP
Active Ras bound to GTP -> downstream signalling
- GAP hydrolyses GTP (releasing Pi) & inactivates Ras
## Footnote
Blockage of GAP activity (mutation) -> constitutive activation of Ras (oncogenic)
155
What are the 3 major pathways that Ras signalling co-ordinates?
MAPK (Raf/MeK/Erk) -> growth
Pi3K -> growth & survival
Ral-GEFs-> regulate cell motility
156
What motility proteins does Ral-GEF signalling inhibit?
RalBP1 inhibits:
Cdc42 -> filopodia formation
Rac -> lamellipodia formation
157
Role of PIP3 in Ras signalling via Pi3K/Akt pathway
It is a secondary mediator required ofr downstream kinases
Formed from series of phosphorylations: PI-> PIP2 -> PIP3 (via Pi3K activity)
158
Structure of Akt for PiP3 binding and the function it induces
AKT has PH domain - pocket for phospholipid to bind.
-> can phos intracellular signalling proteins
- inhibits GSK-3B (inhibitor of proliferation)
- activates HIF-1a (angiogenesis)
- Inhibits Bad (apopoptosis)
- Inhibits TSC2 (protein synthesis inhibitor)
159
PTEN role in Pi3K/Akt signalling
Negative regulator (tumour suppressor)
- converts PIP2 back to PIP2
Phosphatase action - reverses Pi3K action
## Footnote
Phosphatase and Tensin homolog
160
Whats are some effects of Akt/PKB signalling goen wrong?
Anti-apoptotic (increased immortality) -> can inhibit Bad (Bcl-x antagonist) and caspase-9
Proliferative>inhibits GSK-3B, FOXO4, p21 & p27 (stops brakes, reducing inhibition of cell cycle)
Growth -> inhibits Tsc1/2 complex dissociation , mTOR activated which upregulates protein synthesis
161
Describe the structure of Pi3K and hhow it can be compromised?
It has kinase, helical, C2, Ras-binding & p-85a-binding domains -> complex structure
Most (80%) mutations found in helical or kinase domains.
## Footnote
e.g. PTEN -/- histology in epithelium has cancerous growth (lots of PI3K signalling).
162
Role of NF1 gene
Drives turning off of Ras (tumour suppressor)
LoF mutations -> neurofibramatosis type 1(NF1), genetic disorder
- charcterised by benign tumour development in NS
Familial oncogene located on Chr 17
## Footnote
Also associated with malignant peripheral nerve sheath tumours (MPNSTs) and gliomas.
163
Action of function NF1 vs oncogenic mutated version
Neurofibromin binds SPRED1 (sprouty)
-> can then act as Ras-GAP (phosphatase) inhibiting GTP exchange for Ras activation
Removes P from Raf & SH2.
So when mutated -> no negative regulation of Ras signalling
164
Interaction of Ras and Raf in MAPK patwhay
Ras is scaffold for Raf -> lets them (hetero/homo) dimerise.
RAFs then autophos each other + allows phos at other sites
- BRAF only needs one phos in N-region
## Footnote
ARAF, BRAF & CRAF
165
BRAF as a dominant oncogene
Always hyperctivated -> only takes 1 mutation
- Y absence in N-terminal region removes regulation
Mutated in 50–70% of malignant melanomas, 40% of thyroid carcinomas, 30% of ovarian tumours, and nearly 100% of hairy cell leukaemias.
166
What are common BRAF oncogenic mutations?
V600E-BRAF accounting for 80–90% of mutations in melanoma
- allow BRAF to adopt active kinase formaton despite no dimerisation
## Footnote
Mutants signal as RAs independent monomers
167
Stepwise evolution of melanoma
Clear progression model - MAPK & Pi3K pathways need to be activated
NRAS, BRAF, PTEN & AKT sponatenous mutations all contribute to metastasis of cancer cells
168
Karyotyping
Used to count chrom number - polyploidy common in cancer.
Genome toxicity -> results in apoptosis
169
Translocations in chromosomes
Reciprocal - non-homologous chromosomes swap segments
Robertsonian - 2 acrocentric chroms fuse near centromere
170
S-phase generated translocation
DNA Pol jumps from one strand to a homologous strand -> translocation of nucleotide segment.
- Can cause genomic amplification, multiple copies/duplication of mutant
171
Translocations for cancer
Can relocate a proto-oncogene next to a promoter region
e.g. translocation of Myc from Chr8->14, now downstream of constitutively active REGig promoter -> Burkitt lymphoma
Can form a hybrid gene.
Translocation of Bcr (Chr22) & Abl (Chr9) -> fusion forms hybrid oncogene on Philadephia chromosome, permanent PK activity -> CML
## Footnote
Chronic Myelogenous Leaukemia
172
What is senescence?
Cells enter a state of permanent growth arrest without undergoing death
173
True or false: senescence is a normal end for stromal cells
True
Cancer does not undergo uncontrolled growth BUT there is lots of attrition through death, senescence, immune surveillance, insufficient nutrient availability.
As we age, reproductive ability in tissues decreases -> skin thins, muscles weaken, slower immune system.
174
What molecules accumulate w/ time in normal cells?
Cell cycle inhibitors
- can be overcome w/ stimulation of the rigth pathways
175
Role Ki67 and p16 in studying cells
Ki67 tags actively dividing cells
p16 is marker of senescence
## Footnote
Normal cell stop growing after repeated divisions. Senescent cells become 'flat', altered metabolism + inflammatory (morphological changes).
176
What is a telomere and how does it change over time?
6 nuceleotide palindromic sequence -> TTAGGG/AATCCC
G rich & C rich strand
Reduce in size over time + growth rate slows, normal length ~8-10kbps
Also has single stranded 3' overhang of G-rich telomeric DNA
177
How is a T loop formed?
Mismatch on G rich strand -> does not bind C-rich strand, forms D loop
3' overhanging end loops round (T loop) + base pairs to unbound C-rich nucleotide sequence
## Footnote
Prevents DNase cleaving ssDNA, no cellular senescence
178
How is the T/D loop structure (knot) stabilised?
Protein complex - TRF1/2, TIN2, TPP1, POT1 -> lariat structure that protects chromosome ends
POT1 can bind D-loop ssDNA
BUT high resolution fluorescence imaging -> TRF2-dependent t-loop formation, POT1a/b present but not required
(Doksani et al, 2013)
## Footnote
TRF2 is a shelterin protein
179
How is a ssDNA 3' overhang formed during Okazaki fragment synthesis of lagging strand?
180
Describe ssDNA replication of the lagging strand. How does it form telomeric ends?
Primase synthesises short RNA primer
DNA Pol III extends primer into Okazaki fragment
DNA Pol I can displace RNA primer in the way (5’->3’ exonuclease activity) + add bases
DNA ligase joins adjacent nucleotides w/ phosphodiester bond
## Footnote
When primers removed at end of segment -> there is an overhang where telomere can form
181
What is TERT?
Telomerase holoenzyme
- hTERT catalytic subunit
- hTR RNA subunit (primer function)
Holoenzyme because it requires nucleotides to function - not just protein.
## Footnote
Generates telomers at DNA strand ends
182
How does TERT generate telomeric endings?
Attaches to the 3ʹ end of the G-rich strand overhang via H-binding of hTR to the last five nucleotides of the G-rich strand.
Rev transcription of hTR sequences -> extends G rich strand by 6 nucleotides
## Footnote
Can repeat -> 1000s bps
183
What cell is TERT expression present in?
Stem cells
- TERT overexpression in normal cells extends telomeres & stops senescence (allows outgrowth) -> immortal cell characteristic
- Shown in HEK cells w/ and w/o hTERT
184
How does a cell become senescent?
Cell physiological stress induces either telomerase OR p16 (indirectly activates pRB) -> senescence
DNA damage signal induces p53 mediated senescence
## Footnote
pRb & p53 can be inhibited by SV40 LT
185
How is a DNA damage signal generated?
ATM is a homodimer - autophosphorylates before activating Chk2 or p53 directly
ATR senses stalled replication forks, forms ATR–ATRIP and proposed to bind Rad17 when ssDNA present
## Footnote
Both are protein kinases
186
How can TERT be targeted in cancer cells?
Inhibition can stop their growth - but no clinical inhibitor in use (theoretical)
High TERT activity is bad - > lots of telomeres, cancer cells are senescent resistant (immortality), high in neuroblastoma + Ewings sarcoma
Inhibitors can induce senescence
187
How can reduced TERT also be oncogenic?
Lack of telomeric protection:
End to end chromatid fusion, cannot be separated -> can result in chromosomal break or further fusion.
Unequal sister chromatids causes non-disjunction (aneuploidy)
188
What can repeated fusion events due to no telomeres cause?
Too many chromosomes
Extremely enlarged chromosomes (indistinguishable from each other, MEGA chromosomes)
189
Prmoter mutations on TERT expression
Can increase it
Present in >80% glioblastomas
190
What is the DNA damage response?
## Footnote
DDR
Signalling network maximises accurate transmission of genome to the next generation - minimises impact.
Sensor proteins recruit mediator proteins which amplify the signal inducing cellular responses ->
repair, chromatin remodelling, gene expression or apoptosis/sensescence
191
Function of ATM sensor kinase
Major upstream DDR kiase activating Chk2/p53 pathway in G1
192
Function of the ATR
Major upstream DDRkinase for DSB repair
- Chk1/Cdc25/Wee1 signalling is S phase & G2
They prime repair via homologous recombination (HR)
193
Homologu recombination for DSB repair
- Ends resectioned, creating 'overhangs'/sticky ends in damaged DNA
- Enables strand invasion so non-damaged strands can serve as template for damaged strands.
- DNA pol extends damaged strand
- Ligase connects it all together, forming Holliday junctions
- Holliday junctions resolved to give rise to repaired DNA
194
What determines type of DNA damage response?
Cell cycle phase -> ATM (G1) vs ATR (S/G2)
## Footnote
Many proteins coordinate complex processes
195
What phases of the cycle do HR, NHEJ and SSB repair occur in?
HR - S/G2, most accurate (uses homologus sequences), BRCA1/2 + Rad51 crucial
NHEJ - G1->G2, fast but potentially mutagenic
SSB repair - primarily G1 but also S/G2 - PARP enzymes crucial
## Footnote
Dedicated repair pathways deal with distinct lesions in a cell-context dependent manner
196
In what 3 ways is DNA damage not always bad?
Meiotic recombination - DNA break repair via HR promotes pairing of homologous paternal & maternal chromosomes
VDJ recombination of Ab variable region - critical for diversification, can target/kill cancer cells (anti-cancer immune response)
Neural plasticity - neuronal activity induces DNA damage, SSB repair by BER can modulate synaptic transmission
197
What can DDR mutations cause?
Death or hereditary syndromes:
ATM mutant -> ataxia telangiectasia
NBS1 -> Nijmegen breakage syndrome
WRN/BLM helicase mutant -> Werner/Bloom syndrome
BRCA genes -> germline mutation carriers (associated w/ breast/ovarian cancers)
## Footnote
All these syndromes have predisposition to cancer types - BRCA mutations.
198
How is DNA damage in cancer a double edged sword?
Increased mutational burden -> oncogene activation + loss of tumour suppressors (tumorigenesis)
Radio-/Chemotherapies cause mutational overload in rapidly proliferating cancer cells -> death
*treatment option too
199
Desrcibe defects in ovarian cancer
Highly heterogenous, not a single cause/mutation
e.g. 8% BRCA1 germline, 11% BRCA1 methylation, 14% cyclin E amplification
200
Somatic vs germline mutations
Germline - present in gamete -> every cell in body, passed on to offspring
Somatic - absent in gamete, occurs at any time (not in germline cells), not passed to offspring
201
How can BRCA deficient ovarian cancer patients be selectively targeted for treatment?
PARP inhibitors e.g. olaparib
Relies on synthetic lethality i.e. need either BRCA & PARP to survive
## Footnote
Paradigm for targeted therpeutics
202
What is synthetic lethality?
Inactivation of individual genes not harmful, when both inactive -> cell dies (only occurs in cancer)
Olaparib - inhibits PARP
Mutation inhibits BRCA1
-> lethal
203
Why are PARP inhibitors synthetically lethal w/ BRCA deficiency?
When dedificient in both, repair pathways (BER, HR & NHEJ) are error prone and induce greater replicatio stress
-> incompatible genomic instability
204
What are the risks associated w/ BRCA carriers?
Increase likelihood of breast & ovarian cancers - carriers frequently undergo preventative surgical removal of reproductive organs.
205
How can BRCA germline mutants be at increased risk of cancer?
Recessive BRCA mutant still has competent HR - PARPi resistant
But if inactvated on 2nd chrom -> cancer cells, BRCA function abrogated
- PARPi sensitive
206
Use of biomarkers to test for clinical treatment responses
Effective in lab but treatment to clinic difficult - must be rapid + reliable.
e.g. RAD51 immunofluorescence test
207
How does the RAD51 immunofluorescence test determine whether PARPi would be relevant treatments?
Immunofluorescence assay for Rad51(red) & geminin (green)
If no Rad51 -> HR deficient + patient would be PARPi sensitive
- HR efficiency proxy
## Footnote
Rapid, reliable + easily extracted (fixed tissue dample, FFPE)
208
How are resistabce mechanisms a mjor challenge to targeted therpeutics?
- Partial reactivation of BRCA genes via reversion mutations (restore parts of ORFs)
- Promoter switching through chromosomal translocation
- Inactivation of genes that cause synthetic viability (genes that limit steps required for HR)
- Underlying mechanisms
209
How can synthetic lethality be screened for in cancer patients?
CRISPR/Cas9 screens used to identify synthetic lethal relationships:
- ATM deficiency common in many cancers + synthetic lethal w/ many different inhibitors of DNA repair enzymes (PARPi, TOP1i, ATRi + POLQi)
210
What are the 4 main tissue types?
Epithelial - protective layer covering bodys surface, lines internal organs + forms glands
Connective - support, protection + structure. Inc bone, cartilage, blood, adipose & fibrous tissue
Muscular tissue - movement -> skeletal, smooth + cardiac
Nervous - communication between different body parts, inc nueorns + glial cells
## Footnote
BUT every organ is different - cell type content, cell arrangement, structure of ECM/stiffness, ECM content, nutrients/pH/O2/sugars/lipids.
211
What makes up a tumour mass apart from cancer cells?
TME - most solid tumours have 35-65% of tumour made of non-cancer cells, in pancreatic cancer can be 90%
## Footnote
Not all changes are mutations
212
Give 4 ways cancer can alter its microenvironment
Fibroblast recruitment
ECM degradation/intravasation
Macrophage polarisation
Immune suppression
213
Why cant we catch cancer?
Not infectious or transmissable
Tumour growth only seen in syngeneic host after tumour cell transplant - due to natural MHC-1 presentation of antigens, strong affinity for non-self antigens.
## Footnote
No tumours in allogeneic host
214
Are cancer vaccines possible?
Potentially
Mouse immunised w/ irradiated tumour cells injected w/ cells of the same tumour -> rejected + tumorigenesis prevented
When mice injceted w/ different tumour cells, its able to grow (specific immune response)
215
How do cells evade antigen detection?
Do not express MHC-I, so do not induce immune response e.g. 50% down regulation MHC-I in prostate cancer
216
Which immune cell types can support or suppress cancer growth?
TAM, MDSC & TIDC - tumour supportive
T , dendritic, M1 & NK cells - tumour suppressive
Balance of supportive & suppressive cells can determine prognosis - some treatments try to shift balance supportive -> suppressive
## Footnote
TAM - tumour associated macrophages
MDSC - myeloid-derived suppressor cells
217
Anti-tumour TAMs
Activated by LPS/IFN-y
Secrete cytokines, chemokines & metalloproteinases (MMP-7,9 & 12).
Induce cell lysis. Produce reactive O/N species.
218
Pro-tumour TAMs
Hypoxia induced.
Produce pro-angiogenic cytokines/enzymes (IL-8, VEGF), immunosuppressive cytokines , tissue factor/uPA, chemokines + wide range of metalloproteinases.
-> M2-like
219
What pro-tumour cytokines are secreted by cancer cells themselves?
IL-10, TGF-B & IL-4
220
List pro-tumour ligands & their function
TGF-B, IL-10: immune suppression
EFG/FGF: cancer cell growth via RTK signalling
VEGF, CXCL8 & IL-8: angiogenesis
221
List anti-tumour ligands & their function
TNF-a/IFN/IL-1b - immune activation
ROS/TNF-a: cancer cell death via apoptosis
## Footnote
Tumour requires inital inflam molecules to develop
222
Natural killer cell function
Both innate & adaptive, have NKG2D receptor
- can respond to stress altered cells (MCA stress signalling protein) & Abs recognising antigens
Cancer downregulates MICA/MICB (kill me signals) - immune evasion
## Footnote
MHC-I expression has natural inhibitory function on NK cells (opposite to T cells) - act as brake
223
How do NK cells mediate cancer cell death?
Perforin/granzyme pathway: secreted which form pores -> destruction, granzymes enter + trigger apoptosis
Fas/FasL: Fas ligand expressed which bind FasRs on target surface -> caspase/apoptosis activation
TRAIL pathway: TRAIL secreted, binds death receptors (DR4/DR5) on target cell -> caspase/apoptosis activation
ADCC: express Fc receptors that bind Fc region on Ab boun to tumour cells, granzymes released -> apoptosis
## Footnote
TRAIL - TNF-related apoptosis-inducing ligand
224
What is the impact of T cell balance in the TME?
Dictates prognosis of patient.
In cancer: increased Tregs (suppress Tc function, fewer inflam immune cells) -> loss of cancer immuno-surveillance, promote suppression of anti-tumour response, promotes cancer progression
In autoimmunity: reduction in Tregs -> loss of homeostasis & peripheral tolerance, loss of immune response regulation to prevent non-specific side effects
## Footnote
Tregs vs CD8+ T cells
225
How does the TME favour Treg production/secretion?
TGF-B secreted - binds receptor & causes Treg differentiation + increased proliferation
IL-22 binds CXCR4 receptor -> same function, acts as inflammatory chemokine
## Footnote
More Tregs -> natural inhibition of CD4/CD8 T cells, so faster tumorigenesis
226
What is the function of CD8+ cells agaisnt cancer?
Can make it easier to activate Tc cells via removal of inhib signal (e.g. anti-PDL1 Ab) -> success in melanoma
- cancer death induced via granzyme/cytokines like IFN
## Footnote
Increased chance of T cell activation by antigen -> increased immuno-surveillance.
227
What mechanisms do cancer cells employ to avoid immune surveillance?
Suppress MHC expression so antigens not presented - CTLs not employed
Overexpress PDL1 to inhibit T-cell/tumour checkpoint
Overexpress Bcl2 family members + avoid apoptosis
Manipulate balance of immune cell types/mols: more M3 macorphages & Tregs, more TGF-B & IL-10
## Footnote
TGF-B induces macrophage polarisation to M2
228
What is the tumour microenvironment?
Heterogeneous collection of infiltrating & resident host cells, secreted factors & ECM.
Largely made up of epithelial, fibroblasts and smooth muscle cells.
229
Where are >80% human cancers originated?
Epithelium - carcinomas
Form epithelia + stromal histologically complex structures
- stromal fibroblasts overexpress PINCH, form boundary around carcinoma
90% non-neoplastic stromal cells in some cancers -> make up most of hard tissue in many tumours
## Footnote
PINCH is adaptor protein that adheres cytoskeleton to integrin contacts
230
How can cells in teh TME be characterised?
Single cell RNA sequencing (scRNA-seq) to collect data + UMAP used to cluster cell types w/ similar gene expression profiles
Allows comparison
231
What makes up the stroma?
Fibroblasts, myofibroblasts, mesenchymal stem cells, enddothelial cells, pericytes, smooth muscle cells, mast cells, monocytes, macrophages, lymphocytes, adipocytes, ECM
232
Describe the anti-tumour microenvironment
NK cells/M1 - TGF-B, cause death
Neutrophils - ROS
DC - TNF, IL-6, activates T cells
Th1/B - activates CD8+ -> death via IFN-y
## Footnote
CD8+ associated w/ psoitive prognosis
233
Fibroblast function + their role in cancer
Secrete growth factors, cytokines, ECM components -> change TME & promote tumour growth
Also create physical barrier around tumour blocking immune cell/drug entry
- can be converted to cancer associated fibroblasts via TGF-B, PDGF (platelet-derived) & FGF2 secretion by cancer/stromal cells -> triggered to produce more ECM + associated w/ poor prognosis
-> heterogeneous collection of cells
234
What is needed for CAF formation?
## Footnote
Cancer associated fibroblasts
FGF2
CAFs also known as myofibroblasts
- produce far more ECM
235
What are the implications of fibroblasts heterogeneity?
Highly variable response to viral oncogene transformation.
Adult eosophageal - limited dysplasia but structure maintained
Fetal eosophageal - lots of dysplasia, loss of tissue integrity
-> fibroblast heterogeneity across different tissues makes specific treatments very difficult
## Footnote
same oncogene triggers different carcinogenic phenotypes
236
How can CAFs be clinically identified?
Histological stains of a-smooth muscle actin (aSMA) -> high aSMA proportion have much poorer prognosis
- Myofibroblasts predict clinical progression of cancer
- Inverse relationship of stromal myofibroblast & survival time
237
What is the extracellular matrix?
Complex network of proteins, glycoproteins, proteoglycans (e.g. fibronectin, laminin, collagen & elastin) -> cross-linked harder structures
Can also sequester growth factors + soluble mols, continuously remodelled by proteases (liberates tethered mols -> localised high concs)
238
Role of the ECM in the TME
Promotes tumour growth & progression, can create physical barrier around tumour.
Glycan modification indicated in cancer progression.
239
How is stromal cell recruitment and ECM generation a rate limitign step in tumorigenesis?
As it progresses, fib-rich stroma replaced w/ myofibroblasts - generate collagen rich desmoplastic stroma
e.g. human mammary ep cells form tumour
- vol increases faster/reliably when matrigel/fibroblasts injected
## Footnote
Matrigel is synthetic ECM from mouse sarcoma -> tumour grows earlier + all injection sites became tumours (efficiency)
When mixed w/ fibroblasts (produce ECM) -> 100% injections became tumours & double vol of tumours
240
Give an example of reciprocal communication between stromal & tumour cells
Mutated KRAS GoF -> hyperactivity, causes cell autonomous signalling & to nearby fibroblasts (non-cell autonomous)
-> fibroblast behaviour changed
## Footnote
Heterotypic signalling ^ - communication between dissimilar cell types
241
How can KRAS hyperactivity (GoF) change fibroblast activity?
colorectal cancer tumour growth promoted by intestinal myofibroblasts
KRAS-mt -> mRNA expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) higher, promoting migration of primary IMFS through ERK/JNK activation
- more CAFs + increased invasion + intravasation
## Footnote
Kawasaki et al, 2017
242
What is the importance of stroma cor cancer development?
- Cell number, type & communication determines TME functional state
- Composition & function of TME affects cancer progression & treatment
- Cancer cells must influence TME cells to promote tumour growth -> increase proliferation, invasion & intravasation
Also provides physiological support via exchange of mitogenic + trophic factors:
- Carcinoma cells release PDGF to recruit & activate stromal cells + stromal cells release IGFs that sustain carcinoma cell survival (reciprocal signalling)
243
Heterotypic signalling in the TME
Important for normal tissues but heightened in tumour cells.
Depends on mitogenic growth factors (HGF, PDGF, TGF-a), growth inhib factors (TGF-B) & trophic factors (IGF-1/2)
Shown w/ immunofluorescence -> localisation of of FGF-10 (ligand) & FGF-R2b (receptor)
244
What cells are invovled in therpeutic targeting of heterotypic interactions?
Endothelial - inhibitors of FGF, EGF, VEGF, Ang2/Tie2 blocking Abs, angiogenesis inhibitors (endostatin, tumstatin)
Fibroblast - HGF/PDGF inhibitors, FAP inhibitors (sibrotuzumab)
Neut/Macr/Mast cells - anti-inflam inhibitors -> NSAIDs like sulindac and celecoxib, TNF-a inhibitors
Lymphatic cells - VEGF inhibitors prevent angiogenesis
## Footnote
NSAIDs - nonsteroidal anti-inflammatory drugs
Fibroblast activation protein (FAP) is a serine protease primarily expressed by activated fibroblasts
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What are 2 key factors that balance tumour growth?
Angiogenesis (blood supply) & senescence (cell dormancy)
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What is the importance of vasculature?
Tissues w/ high metabolic rates have v. high densities of capillaries - most cells can directly contact a capillary e.g. hepatocytes commonly adjacent to sinusoid (blood access)
- Cells need O2/nutrient supply & waste/CO2 removal
## Footnote
Less organised in tumorigenesis so heterotypic signalling needed to sustain tumour growth
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Composition of vasculature
Blood vascular endothelial cells, pericytes & lymphatic endothelial cells
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Vascular endothelial cells
Single layer, line blood vessels - control protein, cell + oxygen passage through tissue.
In tumour:
- express lower adhesion mols -> impaired barrier function
- express higher immune checkpoint mols -> immunsuppression (PD-L1 which prevents T cell activation)
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Pericytes
Surround blood vessels + embedded in basmenet membrane, support permeability/maturation of vasculature.
In tumour:
Impaired interaction w/ endothelium -> leaky & dysfunctional vasculature
Also paracrine interaction/modulation of stromal and cancer cells in TME using GFs.
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Lymphatic endothelial vessels
Walls of lymphatic vessels - lymph ducts allow APCs access to nodes + drain fluid between cells.
- dissemination route for cancer metastasis
They are direct regulators of anti-tumour immunity, can present tumour Ags but also immune checkpoint mols.
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How does distance from capillaries impact tumour cells?
Human melanoma + rat prostate cancer cells -> capillaries stained w/ CD31 (brown)
O2 availability drops ~60-90um, so does ATP, Gluc + pH
- Lac increases (anaerobic metab)
Cells in hypoxic conditions -> up-regulate p53 -> activates apoptosis, necrosis.
- Some cancers can inactivate p53 to overcome hypoxia -> survive
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What is angiogenesis cotrolled by?
VEGF (vascular endothelial growth factor) secreted by tumour cells, sequestered by the ECM (but remains tethered)
- eventual signal transduction after angiogeneic switch -> endothelial formation
## Footnote
VEGF normall tethered to ECM so needs MMP-9 to cleave (angiogeneic switch)
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Angiogenic switch
Cluster of tumour cells acquire ability to induce neoangiogenesis (angiogenic switch) - signal unidentified
- Accompanied by release of matrix metalloproteinase-9 (MMP-9) from recruited inflam cells (mast, macrophage)
- MMP-9 cleaves ECM components, releasing VEGF for paracrine signalling
Pro-angiogenic factors > anti-angiogenic factors (imbalance)
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VEGF signalling for angiogenesis
VEGF-A and its receptor VEGFR-2, which stimulates mitosis, chemotaxis, and morphogenesis of endothelial cells.
- MAPK -> cell proliferation/gene expression
- Pi3K/Akt -> cell survival/proliferation
- PLCy -> regulates vascular permeability
- FAK/paxillin -> cell adhesion/migration
Endothelial progenitor cells recruited near to tumour -> form contacts to create small capillary vessels, increased proliferation.
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VEGF & its production
## Footnote
Vascular endothelial growth factor
Production governed by O2 availability:
- VHL protein + partners sense intracellular O2 tension
- During hypoxia, complex allows HIF-1α & HIF-1β transcription factor accumulation
- HIF-1 drives angiogenesis related gene expression e.g. VEGF
○ VEGF-A & VEGF-B produced (A predominates angiogenesis)
## Footnote
Synthesised by tumour, macrophages or myofibroblasts
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List 3 pro-angiogenic factors
VEGF
PDGF
Angiopioetin 2 - unstable + leaky blood vessels
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List 3 anti-angiogenic factors
Thrombospondin-1 (TSP-1) - downregulated in tumour growth, can cause apoptosis
Endostatin - inhibits endothelial cell proliferation and migration
Angiostatin
## Footnote
Huang et al 2004
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Capillary formation in angiogenesis
Endothelila cells proliferate + deform -> cylindrical cappilary walls
Move towards highest local conc of angiogenic factors (can penetrate existing tissue)
## Footnote
Tumour capillaries 3x wider than normal + often truncated, disorganised structure
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Excess VEGF on capillaries
Excess VEGF -> separation of adjacent endothelial plasma membranes, cause capillary leakiness (allows increased permeability), Evans blue dye shows leaking
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Heterotypic signalling on capillaries
Causes lack of structural support
Localised PDGF conc needed for pericyte recruitment, PDGF-B secreted by end cells + sequestered to ECM
- mt PDGF-B is not retained by ECM + diffuses away
Lack of structural support, no pericytes
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How does angiogenesis/capillary formation complicate drug administration (IV)?
Causes high intra-tumoral hydrostatic pressure:
1. Tumour associated capillaries leak fluid into parenchymal space of the tumour
2. Cancer cell pop expansion -> lymphatic vessel collapse, disrupting fluid drainage within tumour cores
3. PDGF release by carcinoma cells induces stromal fibroblast contraction, squeezing interstitial fluid in TME
## Footnote
IV drugs cannot be trasnferred against pressure gradient, ~20mmHg interstitial spaces of TME vs ~0.4mmHg in normal tissue
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What are some anti-angiogenic therpeutic strategies?
Anti-VEGF mAb used to return irregular tumorigenic vasculature to normal config + reduce interstitial hydrostatic tumour pressure.
Needs to be normalised not overly reduced, otherwise hypoxia -> invasiveness in cancerr cells
- allows enhanced tissue drug absoption for glioblastoma when treated w/ VEGF-R TK domain inhib (axitinib)
## Footnote
dosing & timing of anti-angiogenic agents need optimisation
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What is cellular senescence?
Irreversible form of proliferative arrest, cells remain metabolically active but cannot re-enter cell cycle.
- induced by nutrient deprivation, DNA damage (telomere erosion), organelle damage, oncogene-induced signalling
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What markers characterise cellular senescence?
p16, p21, cycle arrest, SASP, laminin B1, SA-β-gal & yH2AX.
- Particularly CDKIs (p15, 16 & 21)
Also by absence of proliferative marker Ki67.
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What are 3 types of senescence?
1. Replicative - induced after finite number of cell divisions of normal cells under normal conditions -> telomere shortening
2. Stress-induced premature - triggered by high levels of oxidative & genotoxic stresses
3. Oncogene-induced - triggered by oncogene activation, major anti-tumour mechanism
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What is SASP?
Senescence associated secretory phenotype
Fibroblast SASP factor release -> innduces cnacer prolif + invasion
Senescence thought to protect against cancer development BUT may induce it in paracrine manner
## Footnote
context dependent -> whether it protects or drives progression
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How are SASPs generated?
mTOR -> Mk2 -> stabilisation SASP mRNA
Nf-KB/JAK-STAT -> transcriptional activation SASP genes
DNA damage (BRD4 dep enhancer remodelling, H2AJ accumulation, decrease H3K9me2) -> epigenetic regulation SASP genes
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SASP mechanism of action
It reinforces senescence in autocrine manner + alters adjacent cell migration/proliferation in paracrine manner.
Tumour suppressive: CXCL1 (neutrophil rec), CCL2 (Mac rec), IL-6 recruited
Tumour progressive: IL-6 (in later tumour growth) can drive epithelial-mesenchymal transition (EMT) -> more migratory, so invasion into TME (increased metastasis)
, VEGF/TGF-B secreted, chemokines ^ recruit MDSC (decrease T cell surveillance)
## Footnote
MDSC: myeloid-derived suppressor cells
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What is the function of CDK4/6 inhibitors?
Cancer tretament
Prevent phos of Rb so E2F not left to exrpress S phase proteins.
- cell cycle arrest, no Ag presentation, decreased Treg prolif
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What is the issue with CDK4/6 inhibitor treatments?
Mimic function of p16, which is drives senescence. In PDAC, CDK 4/6 inhibitors induce SASP -> increased vascularity & immune response
## Footnote
PDAC - Pancreatic Ductal Adenocarcinoma
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Use of senolytics
Drugs to target senescnet cells - to remove SASP, preventing regrowth of remaining cells after chemotherapy
e.g. fisetin shown to be potent murine senolytic (Yousefzadeh et al, 2018)
-> combination therapy w/ CDK4/6 inhibitors/chenotherpay could be effective
