Block 2 - Intracellular Cancer Hallmarks

Question 1

Describe the general relationship between proliferative and differentiated states and how this relates to cancer cells

Answer 1

Although not a strict dichotomy, proliferation and differentiation are at different ends of a spectrum:

Differentiated cells are “fixed” in terms of signalling, phenotype and other ways, whereas undifferentiated cells are more tolerant of different situations and can bind a wider range of ligands - PLASTICITY

Cancer generally moves cells from a differentiated to a more proliferative state

Question 2

Describe the advantages of a Proliferative State for cancer cells

Answer 2

1. PROLIFERATION - higher capacity for self-renewal and rapid division
2. APOPTOSIS - often resistant to cell death
3. INVASION AND METASTASIS - maintain some properties of migratory embryonic cells
4. PLASTICITY - more adaptable than differentiated cells, so can respond to environmental changes more easily
5. IMMUNE SURVEILLANCE - reduced expression of surface makers, so less recognisable as abnormal/foreign
6. METABOLIC PLASTICITY - can adapt to different nutrient and oxygen conditions

Question 3

Explain why “Signal Integration” is important to the cell cycle

Answer 3

Cells integrate a range of important stimuli (e.g., Differentiation Factors, GFs, Motility Factors, Survival Factors and Nutrients) to make a “rational” decision about whether to divide or enter quiescence, and also to “programme” the cell cycle phases

[Analogy: a bit like a logic chip]

Question 4

Outline the basic principles of the different cell cycle checkpoints

Answer 4

G1 checkpoint (R) - sufficient metabolites and mitogens to enter S?

S-phase checkpoint - DNA synthesis correct and complete?

G2 checkpoint - organelle synthesis and cell size ready for M-phase?

Metaphase checkpoint - chromosomes aligned?

Question 5

Explain how key protein complexes regulate the cell cycle (and name the different complexes for each stage)

Answer 5

CDKs are always present in the cell, but Cyclin levels fluctuate throughout the cell cycle (due to cycles of transcription and proteolysis), allowing active Cyclin-CDK Complexes to form and drive the cell cycle forward

CycD + CDK4/6: up to R point
CycE + CDK2: G1->S
CycA + CDK2: Early S
CycA + CDK1: Late S + G2
CycB + CDK1/CDC2: M-phase

Question 6

Explain what is meant by the “limited window” of growth signals, and the stability of different cell cycle phases

Answer 6

Cells are responsive to mitogenic signals (Growth Factors) only up to the R (restriction) point in late G1, beyond which the cell is committed to division (cell-autonomous program) -> this makes G1 the most stable phase, as if there are no mitogenic signals, the cell will simply remain in this phase

S-phase has the largest metabolic demand, as duplicating the genome is a big task

Remaining in G2 phase for too long is unstable for cells, as having twice the genome becomes genotoxic

Question 7

Explain how transcription of Cyclin D1 is promoted

Answer 7

Mitogenic signalling from multiple sources and many different pathways feed into the transcriptional control of Cyclin D1:

GF -> RTK -> SOS -> RAS -> Raf/MEK/ERK and Ral/Rac/PAK pathways

GF -> HER2/Neu -> Sp1 -> CycD1

Cytokines -> Jak -> STAT

Also Hedgehog, Wnts/ß-Cat, estrogen receptor, and NF-kB pathways all contribute

“Multiple TFs, downstream of multiple different receptors and ligands”

Question 8

Describe how Cyclin D1 levels are regulated BESIDES control of transcription

Answer 8

TRANSLATION:
- Translation of Cyclin D1 mRNA into protein is ALSO regulated by extracellular signals
- Activated RAS signals via PI3K/PIP3/PDK1/Akt/TSC1+2/RhebGTPase -> mTOR!!!
- When mTOR is activated, it phosphorylates and activates eIF4E-BP-1 complex, allowing it to dimerise with, and activate, eIF-4E
- Active eIF-4E translates Cyclin D1 mRNA into protein

PROTEIN STABILITY:
- Glucose Synthase Kinase-3ß (GSK3ß) can phosphorylate Cyclin D1 on T286, causing translocation from nucleus to cytoplasm, as well as rapid ubiquitination and degradation
- RAS -> PI3K -> Akt -| phosphorylates and inhibits GSK-3ß on S9, thus stabilising Cyclin D

Therefore, 3 levels of Cyclin D control: transcription (many TFs), translation (eIF4e) and proteolysis/stability (GSK3)

Question 9

Describe how the G1/S transition point actually works, including the key proteins involved

Answer 9

Normally, Rb inhibits key target genes involved in the transition to S-phase, including Cyclin E

It inhibits these genes both passively (by sequestering E2F and DP) and actively (by recruiting HDAC to inhibit target genes)

When phosphorylated by CycD-CDK4/6 (and later CycE-CDK2), Rb releases E2F, allowing target genes to be transcribed and synthesised, including S-phase genes and Cyclin E which FURTHER phosphorylates Rb

Once Rb is HYPERphosphorylated, the cell can pass the R point, and Rb remains phosphorylated until it is “wiped” (dephosphorylated) at the end of M-phase

Question 10

What are the “brakes” in the cell cycle and how are they regulated?

Answer 10

Cyclin-Dependent Kinase Inhibitors (CDKIs) act as tumour suppressors by inhibiting CDKs

p16/15/18/19 inhibit D-CDK4/6
p57/p27/p21 inhibit the other complexes

These inhibitors are induced by growth suppressors, e.g., TGFß and p53 (when DNA damage)

They are antagonised by oncogenes, e.g., MYC and AKT

When phosphorylated, these inhibitors are ubiquitinated, freeing the C+CDK complex to phosphorylate Rb. However, if conditions change before the R point, more inhibitor can be synthesised, in which case it must be phosphorylated and ubiquitinated AGAIN to progress the cell cycle

Question 11

Describe the role of Cyclin E in Breast Cancer

Answer 11

Elevated levels of cyclin E are associated with much lower survival rates in breast cancer patients, and can cause abnormal mitosis

High Cyclin E levels causes a more aggressive form of cancer, as well as genome instability

Cell cycle genes are a VERY common genetic alteration in cancer, e.g., loss of Rb or p16, or gain of Cyclin D1 and CDK4/6

Question 12

Describe the nature of each main cell cycle checkpoint in terms of DNA

Answer 12

G1: DNA Damage Checkpoint (entry into S blocked if genome damaged)

S: DNA Damage Checkpoint (DNA replication halted if genome damaged)

G2: Entry into M-phase blocked if replication not complete

M-phase: Anaphase blocked if chromatids not properly assembled on mitotic spindle

If any of these checkpoints identify an issue, the cell cycle is delayed, until either repair is complete and the cell cycle can continue, or apoptosis/senescence occur instead

Question 12

Describe how inhibitory and activating signals can be integrated at the same time in the cell cycle

Answer 12

They target different parts of the cell cycle machinery, e.g.:

Inhibitory:
- TFBß binds TGFR, activating p15 (which inhibits D+CDK4/6)
- TGFR also (weakly) activates p21 (which is also activated by DNA damage), which inhibits other C+CDK complexes

Activating:
- Mitogens activate RTK -> PI3K -> Akt/PKB
- Akt/PKB translocates to the cytoplasm and inhibits p21 + p27
- p21 and p27 can no longer inhibit C+CDK complexes

These are just examples, but the G1/S transition is ultimately a balance of many different signals (remember the big slide with no “learn me”), which integrate into one axis (activation/inhibition of Rb)

Question 13

Name as many “GO” and “STOP” signals in the pathways controlling Rb and the R point transition as possible

Answer 13

Wnts (GO) -| APC (STOP) -| ß-catenin (GO) -> D1 -> CDK4/6 -| Rb

Mitogens -> RTK -> RAS -> Raf -> D1

PI3K -> Akt/PKB -> NF-kB -> D1

TGFßR (STOP) -> Smad3/4 -> p15 -| CDK4/6

MYC and BCR-ABL -> D2 -> CDK4/6

FOXP3 -| Cks1/Skp2/Cul1 -| p27 -| E/CDK2

Mitogens -> RTK -> Akt/PKB -| p27 and p21

There’s more if interested :D

Question 14

What are the main reasons why it is useful to monitor cell growth in the context of cancer?

Answer 14

1. DIAGNOSE cancer (look for abnormal growth)
2. MONITOR treatment response (most drugs inhibit cell growth - see if they achieve this)
3. IDENTIFY new drug targets that inhibit growth
4. DEVELOP personalised treatment plans (test patient cells for growth)

Question 15

What are the two types of effects seen in treatments targeting the cell cycle?

Answer 15

CYTOSTATIC - causes arrest in cell cycle, but little or no programmed cell death

CYTOSTATIC - cell cycle EXIT, and programmed cell death or necrosis

In terms of a tumour, cytotoxic causes the tumour to shrink, whereas cytostatic causes the tumour to stay the same size, or grow slowly

Question 16

Name the methods of measuring cell growth/cycle that were mentioned in the lecture, and state the basic principle of each one

Answer 16

1. Simple Cell Count (using crystal violet and hemocytometer)
2. FACs (measure amount of DNA)
3. EdU/BrdU (stains S-phase-Generated new chromosomes to show G1/S transition)
4. Fucci Cells (live cell cycle imaging)
5. Ki67 (shows which cells in a TUMOUR are in active cell cycle, useful for histology and patient samples)

Question 17

Explain the principle of a simple cell count and how useful it is for analysing the cell cycle

Answer 17

Count cells manually with a hemocytomer; stain the cells and quantify with solubility

Very easy to quantify and good for providing an initial idea, but cell number does not exactly reflect the cell cycle, and cells can be lost due to decreased adhesion or lower survival

Question 18

Explain the principle of FACs and how useful it is for analysing the cell cycle

Answer 18

Fluorescent probes (e.g., Propidium Iodide) measure DNA in a stoichiometric manner, as the number of probe molecules which bind to DNA is proportional to the number of chromosomes

On the FACS graph, can distinguish between diploid cells (1X), cells synthesising DNA (1.5X) and cells pre-mitosis (2X) - therefore, can provide a global profile of how many cells are in which stage of the cell cycle, and whether the plot profile is characteristic of replicating cells, cells in G1/S arrest, cells in G2/M arrest, etc.

Note: must remove RNA with RNase, otherwise probes will bind both DNA and RNA

Also: some cells may have LESS than the G1 amount of DNA due to apoptosis or gametes

Question 19

Explain the principle of EdU/BrdU and how useful it is for analysing the cell cycle

Answer 19

BrdU and EdU are fluorescent dyes which can incorporate into, and stain, newly generated chromosomes, thereby labelling any cells which have recently entered S-phase (within the last hour or so)

This is highly quantitative, but requires cells to be fixed (therefore only providing a snapshot) and, unlike FACS, doesn’t allow all stages of the cell cycle to be seen, only S-Phase

Question 20

Name the four conditions/drugs mentioned in the FACS examples, explain how each one would affect a FACS plot, and why

Answer 20

PALBOCICLIB -> expect the middle graph (G1/S arrest, as CDK4/6 is inhibited)

PACLITAXEL -> expect third graph (G2/M arrest, as MTs involved in segregation are inhibited)

If remove all mitogens from a cell with mutant RAS -> expect third graph (as mutant RAS pushes cells past R point regardless of mitogens)

FLAVOPIRIDOL -> probably third result, but hard to be sure because different cell lines have different responses to non-selective CDKIs, depending on how the cell line is dysregulated (e.g., Rb, Cyclin D, etc.)

Question 21

Do CDKIs induce cell death?

Answer 21

Usually, a cell can survive for a certain amount of time in a particular phase, but eventually will have to make a fate decision, either progress or apoptosis - therefore, eventually, CDKIs can lead to cell death

Question 22

Given the mentioned effects of CDKIs on cells, how are they usually used as treatments?

Answer 22

Most CDKIs are not used individually as treatments, but in combination with something else, e.g., a cytotoxin (so that cells grow less AND die more)

KEY EXAMPLE: BREAST CANCER
- CDK4/6 inhibitors used in combination with Tamoxifen
- Tamoxifen binds Estrogen Receptor, and prevents Estrogen from activating it
- This prevents E2-ERa complex from upregulating cyclin D
- Since both Cyclin D and CDK4/6 are inhibited/downregulated, cell cycle progression is significantly hindered

Can also use aromatase inhibitors (degrade E2?) or Fulvestranz? (degrade ERa?)

Question 23

Explain the principle of Fucci Cells and how useful they are for analysing the cell cycle

Answer 23

In Fucci Cells, we tag proteins, which would normally be degraded in a particular stage of the cell cycle, with different colours (this staining can be transient or stable, and we can even produce entire organisms consisting of Fucci cells)

By making use of stability changes of cell cycle-dependent proteins, this staining can be used to gain live or snap shots of which cells are in G1/0, S and G2/M

These cells can also be grown in an approximately tumour-like formation (sphere) and observe WHERE in the tumour proliferation takes place

PROS:
- Live imaging
- Complex-model-compatible

CONS:
- Hard to quantify, unless with FACs
- Not all cell stains are compatible with human samples
- Need cell lines that actively divide well

Question 24

Explain the principle of Ki67 and how useful it is for analysing the cell cycle

Answer 24

In human tumour samples, we can’t do FACS analysis without destroying the tumour structure - instead, we can use Ki67 to determine whether cells are in Active Cell Cycle or G0

Ki67 protein is expressed during all active stages, and is involved in various processes related to division, e.g., chromosome condensation

• Only a snapshot, but good for histology and patient samples
• Cannot identify WHICH stage of cell cycle, only whether active or not

Question 25

What are the two types of phosphorylation associated with RTKs?

Answer 25

1 Auto-transphosphorylation of the receptor upon dimerisation [both initial and secondary transphosphorylation required for fully active receptor]

2. Cross-phosphorylation of adaptor kinases (binding partners of the receptor), e.g., Tyk2 and Jak1 - binding partners of a-interferon receptors

Question 26

What are the most common causes of oncogenic RTK signalling?

Answer 26

Constitutive activation due to mutations affecting structure (e.g., loss of extracellular or regulatory domain, mutations in key residues)

Overexpression: either genomic amplification, or transcriptional upregulation (e.g., Myc mutations) leading to hyperactivation (more receptor, so harder to turn off signalling, but still requires ligands)

Of these, mutations are MORE common

Question 27

How common are RTK mutations in cancer?

Answer 27

Depending on the type of cancer, they can be VERY common:

• EGFR mutations in 10-15% of non-small cell Lung Cancers
• EGFRvIII mutations in up to 50% of glioblastomas

EGFR overexpression is commonly found in tumours such as lung, breast, head, neck, etc., as epithelial cells naturally respond to, and express, EGFR

Question 28

What is paracrine signalling and what are some key examples?

Answer 28

In normal physiology, paracrine signalling is cell-to-cell communication acting locally on neighbouring cells to co-ordinate cellular responses within tissues and organs, e.g., prol/diff/survival

Examples:
- Neurotransmitters (nervous system)
- Growth factors (blood vessel growth)
- Cytokines (immune cell activation)

Question 29

In what way is RTK signalling supposed to be restricted, and what can be the result if this goes wrong?

Answer 29

RTK signalling is meant to be tissue- and ligand-restricted

Cancer cells often express receptors NOT associated with their tissue of origin - this is one of the most common dysfunctional phenotypes of cancer

RTK signalling can ALSO drive autocrine signalling:
- Cancer cells can produce and respond to their own ligands (e.g., EGFR -> RAS -> TGFa synthesis -> EGFR)

It can be hard to quantify how common this is in tumours, as it is difficult to tell in vivo whether a ligand is from the cancer or TME; easier to see in vitro when cell lines are isolated

Note: many of the ligands mentioned in the big table (e.g., HGF, IGF2, IL6/8, PDGF, TGF, VEGF, etc.) can signal to OTHER cancer cells, clones in a polyclonal tumour, OR to the TME including co-opting stromal cells [NOT JUST AUTOCRINE]

Question 30

Describe the extent to which RTKs share structure with each other

Answer 30

Different families of RTKs have different architecture, but share domains:

• All share the tyrosine kinase domain (even non-receptor tyrosine kinases, e.g., SRC)
• Extracellular domains may interact with different factors
• EGFR has TWO Cys-rich domains, IGF1R has cleaved ECDs joined by disulfide bridges, several have Ig-like domains, etc.

Question 31

What do some RTKs need in addition to their ligand in order to activate?

Answer 31

COFACTORS (e.g., Heparin is a necessary cofactor for FGF to fully activate)

This can help fine-tune where in the body receptors are actually responsive (e.g., only when properly polarised in an epithelial sheet)

Question 32

What is one (mentioned + highlighted) example of how RTK signalling can go wrong?

Answer 32

GENE FUSIONS:
If a fusion protein is formed, containing a receptor protein fused to a ligand partner, then dimerisation (and downstream signalling) will constantly occur, even in the absence of the signal molecule

Examples:
- Ros(R) + Fig
- PDGFR + BCR
- FGFR + FIM

etc.

Question 33

How diverse are the adaptor proteins that can dock with activated RTKs?

Answer 33

Wide range, not just canonical pathways (MAPK + PI3K), but also many other internal molecules [e.g., EGF adaptors: PLC-y, Shc, Cbl, JAK2, Grb3, SHP1]

However, ALL have SH2 and SH3 (key domains)

Question 34

Describe how the structure of Src relates to its activation

Answer 34

Normally, Src is inactive: the SH2 domain is held in place by a phospho-Tyrosine in its own structure, while SH3 is held by its own linker segment, creating a closed structure for the overall protein, in which the position activation loop blocks the activity of the catalytic cleft

When an RTK (e.g., PDGFR) is activated, it provides a Pro-rich domain for SH3 to bind to and a new phospho-Y for the SH2 domain, which causes a structural rearrangement of the Src protein

The Y416 residue of the activation loop can now be phosphorylated, which exposes the catalytic cleft and fully activates Src

Note: activation can be via self-phosphorylation, RTKs such as EGFR, or other adaptor kinases such as FAK

Question 35

Describe the TWO way in which RAS can be activated by adaptors

Answer 35

Either:

RTK -> Grb2 -> SOS (GEF) -> RAS
[SH2 of Grb2 docks on pY, then SH3 recruits SOS via SH3]

RTK -> Shc -> Grb2 -> SOS -> RAS
[SH2 of Shc binds pY, then pY of Shc recruits SH2 of Grb2…]

Question 36

Summarise the main pathways activated downstream of RAS

Answer 36

RAS is a signalling node which activates 3 CANONICAL signalling pathways:

1. RAS -> PI3K -> PIP3 -> RhoGEFs and also Akt/PKB -> Bad, mTOR, GSK3ß, FOXO
2. RAS -> Raf -> MEK -> Erk1/2 -> [Msk1, RSK, Mnk1, Ets, Elk1, SAP1]
3. RAS -> Ral-GEFs -> RalA and RalB -> Sec5, Exo84, RalBP1 (inhibits Cdc42 and Rac)

Question 37

Describe the function of PI3K

Answer 37

PI3Ks phosphorylate lipids, not proteins

Key example:
Phosphorylates phosphatidylinositol (PI) to PIP2, then to PIP3, a key secondary mediator required for activation of downstream kinases

Note: PIP2 can also be converted to IP3 by PLC

Question 38

Explain the significance of PIP3 in one of the major signalling pathways downstream of RAS

Answer 38

PIP2 <-> PIP3 is a reversible reaction (PI3K produces PIP3, PTEN produces PIP2 - this makes PTEN a negative regulator and TSG)

When PIP2 converted to PIP3, it recruits the kinase Akt/PKB via its PH domain

This kinase is then phosphorylated and activated, and goes on to activate HIF1a (angiogenesis), and inhibit GSK3ß (a proliferation inhibitor), Bad (a pro-apoptotic protein) and TSC2 (a protein synthesis inhibitor), among many other pathways related to survival/proliferation/differentiation

Also notable: Akt inhibits p21 and p27, casp9, and activates Mdm2 (anti-apoptotic protein)

Question 39

Describe the most common PI3K mutations and their significance

Answer 39

Most commonly mutated in helical or kinase domains

Oncogenic mutations drive survival and cell cycle (tissue scans show effect of high pAkt on cell morphology and behaviour

Question 40

What is NF1 and what is its significance in cancer?

Answer 40

Nf1 is a GAP which inactivates RAS (making it a TSG)

Normally, NF1 inactivates RAS via GTP hydrolysis when no RTK signalling is present, but is phosphorylated and degraded by active RTK

However, LoF mutations in NF1 gene lead to Neurofibromatosis type 1, a genetic disorder which involves benign tumours of the nervous system (neurofibromas)

Also associated with malignant peripheral nerve sheath tumours (MPNSTs) and gliomas

Question 41

What is Sprouty and what is its relevance to cancer?

Answer 41

Sprouty proteins are a conserved family of MAPK/ERK negative regulators, which inhibit activation of ERK in response to several trophic factors

Loss or dysregulation of Sprouty has been associated with cancer

Question 42

Describe the significance of RAF and mutations thereof in cancer

Answer 42

RAFs are activated by RAS (which acts as a scaffold) and are involved in the MAPK signalling pathway

RAFs can form homo- OR heterodimers (e.g., ARAF-BRAF, BRAF-BRAF, etc.), which allows autophosphorylation each other, and phosphorylation at other sites

BRAF is the most easily mutated RAF due to differences in the N-region -> by far the most common BRAF mutation is V600E, which accounts for 80-90% of mutations in melanomas

V600 mutations allow BRAF to adopt an active kinase conformation, even in the absence of dimerisation -> RAS-independent monomers

Question 43

[Not a question - free 5/5]

Reminder that RAF, RAS, Akt mutations, etc., all relate to the stepwise model of cancer formation

A mutation in one may lead to a benign legion, while other mutations are required for early malignant neoplasia, and late malignant neoplasia, and finally a transformed cancer

Basically just remember to relate essays to this model

Answer 43

Cool beans

Question 44

What are the 3 broad routes by which RTK signalling may be targeted therapeutically?

Answer 44

1. Use monoclonal antibodies (mAbs) to block ligand binding
2. Use mAbs to prevent receptor dimerisation
3. Use drugs that inhibit kinase activity (TKIs)

Question 45

Why might targeting the LIGANDS of RTKs be ineffective in a tumour?

Answer 45

If the RTKs have an activating mutation, then mAbs against the ligand will have no effect, as the mutation is downstream of them

Also, in a tumour (especially a polyclonal tumour) there may be many ligands, or one ligand may bind multiple receptors, and a single mAb cannot target all of these interactions

Question 46

Discuss the routes of RTK targeting in the context of the EGFR specifically

Answer 46

Context: EGFR has multiple ligands/agonists (e.g., EGF, BTC, amphiregulin, TGFa, etc.), so more effective to target receptor than ligands

There are mAbs which block receptor dimerisation (Cetuximab, Panitumumab)

There are ErbB Family Kinase Inhibitors (Lapatinib, Afatinib, Neratinib)

There are EGFR-specific kinase inhibitors (Erlotinib, Gefitinib)

And there is a T790M mutant-specific inhibitor (Osimertinib) which is commonly use in relapse patients

Note: these are usually used in conjunction with other therapies nowadays, as tumours tend to develop resistance

Question 47

Explain the difference between “Progression-Free Survival” and “Overall Survival”

Answer 47

“Progression-Free Survival” measures the time following a particular treatment during which the disease does not progress

“Overall Survival” measures the time between receiving therapy and succumbing to illness

Progression-free survival is often used as it can be recorded earlier, thereby providing quicker information on how well a therapy is working

Overall survival is also impacted by other factors such as additional illnesses

Question 48

What has repeatedly been found about responses to RAF and MEK inhibitors as monotherapies?

Answer 48

If RAF or MEK are inhibited, but RAS is still being activated by external ligands, RAS usually finds a way to re-activate AKT and ERK

In this way, the TME elicits innate resistance to RAF inhibitors

Mutant specific therapies (e.g., Vemurafenib/Dabrafenib target BRAFV600E) or MEKi monotherapies (e.g., Trametinib) have good initial responses, but relapse is very common

Note: PI3K/AKT inhibition has had less success as monotherapies

Question 49

Is Combination therapy more successful than BRAF or MEK monotherapy?

Answer 49

Yes - combining BRAF and MEK inhibitors has become Standard Combination Therapy (e.g., Dabrafenib plus Trametinib is more successful than Vemurafenib alone)

Question 50

Is it possible to inhibit RAS rather than RAF or MEK?

Answer 50

It is very difficult to develop compounds that stop GTP binding for RAS specifically, without having off-target effects on other GTPases

Some evidence that it may be possible (future BR?)

Question 51

What is meant by Spectral Karyotyping (SKY) analysis?

Answer 51

SKY analysis is a Fluorescence in situ Hybridisation (FISH) technique in which fluorescent probes are used to ‘paint’ chromosomes, enabling better identification of aberrant chromosomes than is possible through traditional banding techniques

Question 52

How and when can chromosome translocations occur?

Answer 52

1. In mitosis: mitotic recombination allows part of an arm to be exchanged between chromosomes, which can lead to a loss of heterozygosity in daughter cells (depending on how those chromosomes separate in mitosis)
2. In S-phase: if DNA polymerase erroneously switches from one chromosome to the homologous chromosome, then back again, a translocation of genetic information can occur

Question 53

What is senescence?

Answer 53

Senescence is the result of cellular aging: after a certain number of divisions, normal stromal cells stop proliferating and undergo phenotypic changes (e.g., become flat, have altered metabolism, enlarged nuclei, loss of structure, inflammatory, etc.)

This is partly due to accumulation of Cyclin-CDK Inhibitors (e.g., p15/16/18/18 and p27/57/21) - growing cells in a special medium can overcome this accumulation

Question 54

Which body cells do not undergo senescence and why?

Answer 54

STEM CELLS - cells in these niches have a degree of immortality, partly due to maintenance of their telomeres

Question 55

What are telomeres and how does their function relate to cancer?

Answer 55

Telomeres are repetitive sections of DNA which cap the ends of linear chromosomes, thereby protecting against chromosome shortening

Telomeres reduce in length over time due to DNase activity on ssDNA (which correlates with the decrease in growth rate of cells), and act like a ‘molecular clock’ counting down to determine whether a cell still has the capacity to divide

When telomeres get so short that critical DNA is affected, damage signals are released, leading to p53 overexpression, while cell stress signalling induces p16 (a CDKI) - altogether, this promotes senescence in normal cells

If telomeres are lost but senescence does NOT occur, unprotected chromosome ends can undergo end-to-end fusions, preventing segregation in mitosis, and leading to anaphase bridges, new points of fusion, and repeated break-and-fusion events, including with non-homologous chromosomes, leading to genome instability

This increases the likelihood of oncogenic events due to loss of regulatory domains in oncogenes, or loss of tumour suppressors; it can even lead to MEGA chromosomes - a complete loss of control of gene number

TERT overexpression (i.e., telomere maintenance) is required in some cancers, e.g., neuroblastoma and Ewing’s sarcoma

Question 56

Describe the molecular structure of the telomeres at the ends of chromosomes

Answer 56

Telomeric DNA appears as a “T-loop” due to the overhanging 3’ end of the G-rich strand:

A displacement loop (or D-loop) in the G-rich strand allows the 3’ overhanging end of the same (G-rich) strand to interact with the C-rich strand, thereby tying the T-loop in place

This shape is maintained by a range of key proteins:
- TRF1 and TRF2 bind the repetitive dsDNA region, while POT1 binds the 3’ overhang, with other proteins (TPP1 and TIN2) linking these proteins together to secure the T-loop structure

Question 57

What feature of DNA replications makes telomeres necessary?

Answer 57

When replicating the lagging (3’) strand of DNA, Okazaki fragments must be produced, where a 35bp RNA primer is placed, a small fragment of DNA replicated, then the primer removed and a more distal one produced as the replication fork moves

At the end of the chromosome, there is an overhang where the primer is removed, and this is where the generation of ssDNA telomeres are necessary for replication to be completed

Question 58

How are telomeres generated?

Answer 58

The human telomerase holoenzyme catalyses telomere formation:

The holoenzyme attaches to the 3’ end of the G-rich strand overhang via hydrogen bonding of the hTR RNA subunit to the last 5 nucleotides of the G-rich strand

Then, the hTERT catalytic subunit catalyses the extension of the G-rich strand by six nucleotides, via reverse transcription of sequences in the hTR subunit

This is repeated for thousands of bases

Question 59

What is unusual about the types of mutations that often lead to TERT overexpression?

Answer 59

It is often caused by mutations in PROMOTERS, rather than coding DNA (for example, by allowing binding of different TFs to the promoter) - especially common in glioblastomas, myxoid liposarcomas, and oligodendrogliomas

Non-coding mutations are unusual for an oncogenic event

Question 60

Is TERT inhibition a potential treatment?

Answer 60

TERT inhibition could potentially inhibit tumour growth, but would have negative side effects on aging, so is unlikely to be widely used

Question 61

State the advantages of Genomic Instability for cancer cells

Answer 61

1. More activation/amplification of oncogenes and loss of TSGs
2. Gene fusions (e.g., BCR-ABL and other RTKs)
3. Altered signalling pathways (e.g., kinases or TFs)
4. Increased genetic diversity -> heterogeneity and polyclonal tumours
5. Accelerated evolution (quick adaptation allows cells to grow and survive)
6. Resistance to therapy (due to more genetic alterations)
7. Immune evasion (loss of expression of genes such as tumour antigens)
8. Modulation of TME (overexpression of VEGF, stroma-changing ligands e.g., TGF-ß, etc.)

Note: Genomic Instability is greatly increased by loss of telomeres, as sticky ends lead to fusions and breaks

Question 62

State which stage of mitosis has a key checkpoint which can be dysregulated in cancer, and summarise how this checkpoint works

Answer 62

METAPHASE (Spindle Assembly Checkpoint, SAC):

The checkpoint prevents mitosis from progressing until ALL chromosomes are properly attached to the mitotic spindle

Once chromosomes are attached, the Mitotic Checkpoint Complex (MCC) binds to Cdc20, which then activates APC, prompting it to rapidly degrade cyclin B (thereby inactivating CDK1 to promote mitotic exit) and securin (releasing separase to separate sister chromatids, leading to anaphase)

Question 63

What is meant by “mitosis is a race” and how can this be used for therapeutic targeting?

Answer 63

It is a race between Division (promoted by falling Cyclin B levels) and Cell Death (promoted by damage signal accumulation due to the lack of nucleus, limited transcription and protein synthesis, etc.)

If damage signals accumulate faster than Cyclin B levels fall, then the cell may undergo apoptosis

Targeting mitosis aims to stall cells for long enough that the damage threshold can be reached and prompt apoptosis

Question 64

Describe the different types of microtubule inhibitors and their potential use in cancer therapy?

Answer 64

Principle: target microtubules required for chromosome segregation, thereby stalling mitosis to induce cell death [note that this also targets cells in normal tissues which divide rapidly, e.g., hair follices, GI tract, blood production]

VINCA ALKALOIDS (e.g., Vinblastine, Vincristine) prevent the formation of stable microtubules

TAXANES (e.g., paclitaxel/taxol, docetaxel - all alkaloid derivatives) prevent microtubule depolymerisation

Question 65

Explain how cells treated with taxanes (e.g., taxol) appear on a FACs plot

Answer 65

Similar to G2/M arrest cells -> highest peak is at 2X DNA content

However, also peaks for multinucleated cells (e.g., 4N, 8N, etc.) as cells may continue to attempt division but fail to separate the chromosomes -> genotoxicity can lead to cell death, either immediately or after a few rounds of aberrant division

Question 66

Summary of final lecture:
- Targeting mitosis: attempt to disrupt division to promote cell death
- Cancer cells can “push back finish line” for cell death in mitosis
- More survival signalling activated -> more resistance to CDKIs
- Genomic instability is a potent advantage for developing and treated tumours

Answer 66

Cool Beans

Question 67

Name 5 common sources of DNA damage (i.e., DNA damaging agents)

Answer 67

• DNA replication stress
• Reactive oxygen species (ROS)
• Ionising or UV radiation
• Chemotherapies
• Environmental chemicals

Question 68

State the distinct types of DNA legions

Answer 68

• Base mismatches
• Single-strand breaks (SSBs) + abasic sites
• Adducts
• Intrastrand crosslinks
• Double-strand breaks (DSBs)
• Interstrand crosslinks

Question 69

State the main types of DNA repair

Answer 69

• Mismatch repair (MMR) for base mismatches
• Base excision repair (BER) for SSBs
• Nucleotide excision repair (NER) for adducts and intrastrand crosslinks
• Homologous repair (HR), and Non-Homologous End Joining (NHEJ) for DSBs

Question 70

Explain why the DNA Damage Response (DDR) is more complicated than simply the types of repair

Answer 70

The DDR involves not only DNA repair, but also the recruitment of mediators by DNA damage sensor proteins, which leads to signal amplification to induce responses such as cell cycle arrest, chromatin changes (making DNA accessible to repair enzymes), gene expression and apoptosis/senescence [Essentially, the DDR maximises accurate transmission of the genome to the next generation]

ALSO, not all repair happens at the same time:
- Different DSB repair pathways are activated in different cell cycle stages (e.g., S and G2 need a different pathway due to the presence of sister chromatids)
- In G1, a major upstream DDR kinase called ATM activates the CHK2/p53 pathway, leading to inhibition of CyclinA/E and CDK2
- In G2, a different major upstream DDR kinase activates CHK1/CDC25/WEE1 signalling, leading to homologous recombination (HR) - requires sister chromatid [This pathway also leads to inhibition of Cyclin A/B and CDK1/2]

Overall, hundreds of different proteins are involved in co-ordinating the most appropriate DDR pathway depending on point in the cell cycle, and the chromatin environment/architecture -> extremely complicated

Extremely broad summary:
- PARP enzymes aim to repair SSBs
- NHEJ for DSBs in G1, S and G2
- HR in S and G2 only -> most accurate, requires homologous sequence and BRCA1/2, RAD51 etc

Question 71

Name three examples of useful/important DNA damage

Answer 71

1. Meiotic Recombination
2. VDJ Recombination in the immune system
3. Neural plasticity

Question 72

Explain why DNA “Damage” is important in meiosis

Answer 72

In order for meiotic recombination and accurate segregation to occur, the formation of a chiasma, and DNA break repair via HR, and required

Question 73

Explain why DNA “Damage” is important for the Immune System

Answer 73

VDJ Recombination (i.e., recombination of the V, D and J segments that contribute to antibody diversity) is vital in achieving diversity of antibody specificity during B cell development in bone marrow

Here, induction of DSBs and repair thereof allow random arrangement of variable (V), diverse (D) and joining (J) genes that make up the antigen-recognising region of antibodies, contributing to a well-developed immune system that can respond to a wide range of antigens/pathogens

ALSO Class Switch Recombination:
Different classes of antibodies are specialised for different stages of the immune response, but all classes contain the same primary sequence - certain sequences must be “looped out” via DNA breaks and repair, in order to switch and produce the different classes
-> This is vital for a well-coordinated course of events in immune function

Question 74

Explain why DNA repair is important for neural function

Answer 74

SSB repair by BER modulates synaptic transmission

DNA repair modulates neural plasticity, altering synaptic connections and transmission

Question 75

Explain how important the DDR is for human health, and what can happen if it is dysfunctional

Answer 75

Extremely important:
- Mutations in many DDR genes are lethal
- Others cause hereditary syndromes, e.g. mutations in:
- Apical kinases, such as ATM/DNA-PKcs lead to Ataxia telangiectasia
- FANC genes lead to Fanconi anemia
- NBS1 -> Nijmegen breakage syndrome
- WRN helicase -> Werner Syndrome
- BLM helicase -> Bloom syndrome
- BRCA genes -> Predisposition to cancer

The common feature of all of these is a predisposition to cancer

Question 76

Why is DNA damage in cancer described as a “double-edged sword”?

Answer 76

On the one hand, DNA damage can promote tumourigenesis via oncogene activation and loss of TSGs

On the other hand, the genomic instability of rapidly proliferating cancer cells leads to increased mutational burden, which can be targeted by radio- or chemotherapies which aim to tip the balance and induce cell death due to mutational overload

Several conventional chemotherapies target replication stress (but many also affect normal proliferating cells, and thus have severe side effects)

Question 77

What are the three broad areas to consider when trying to develop targeted DNA damage cancer therapies with LESS side effects?

Answer 77

1. What makes cancer cells different from normal proliferating cells?
2. How can these differences be selectively targeted without affecting normal cells?
3. How can we identify (stratify) patients that will respond well to these treatments?

Question 78

What is the general message of the “What makes cancer cells different” segment?

Answer 78

Although cancer is extremely heterogeneous, and can involve a wide range of mutations, it is possible to group cancers by TYPES of mutations they have (e.g., around 40% of serious ovarian cancers have mutation in either BRCA1 or BRCA2)

Question 79

How can the “differences” in cancer cells be targeted selectively without affecting normal cells (in the context of DNA damage and repair)?

Answer 79

Using our understanding of synthetic lethality, we can use PARP inhibitors to target only cancer cells with BRCA1/2 mutations (e.g., using olaparib/Lynparza)

This is because these two pathways are synthetically lethal - inhibition/inactivation of either pathway alone is not lethal to cells, but when BOTH are inactive, the cell dies

In the case of PARP inhibitors, normal cells are not harmed by the drug as they still have functional BRCA1/2 genes, however, cancer cells now have neither pathway functional and are thus prone to cell death

More mechanistic detail:
- PARP repairs SSBs before they can develop into DSBs
- If PARP1 is inactivated (by an inhibitor such as olaparib), SSBs cannot be repaired and become DBSs
- In normal cells, functional BRCA1/2 is able to repair these increased DSBs via HR
- However, in cells where BRCA1/2 is inactivated via mutation, HR cannot take place for these many DSBs, meaning NHEJ or Alt-EJ must be used, which are mutagenic, and lead to genomic instability and cell death

Thus, there are minimal side effects in normal cells

Question 80

Given the information in Lecture 8 surrounding Synthetic Lethality, how can we stratify patients that will respond to the identified treatments?

Answer 80

BRCA1/2 can be used as a biomarker to stratify patients for PARP inhibitor (e.g., olaparib) treatment
-> HOWEVER, other genes can also lead to HR deficiency, and BRCA gene inactivation can occur via EPIGENETIC means such as silencing via promoter hypermethylation
-> This make biomarkers more complicated

RAD51 is a possible biomarker:
- RAD51 is recruited to DNA damage sites, and can be used as a proxy for cells successfully performing HR
- An immunofluorescence assay for RAD51 can reveal HR deficiency, and suggest that patients may be sensitive to PARP inhibitors or platinum-based drugs

Question 81

Why are resistance mechanisms such a major challenge for PARP inhibitors as a cancer therapy?

Answer 81

• Partial reactivation of BRCA genes via reversion mutations (which can restore parts of the ORF)
• Promoter switching through chromosomal translocation (affects BRCA genes inactivated via promoter hypermethylation)
• Inactivation of genes that cause synthetic VIABILITY (e.g., genes that limit steps required for HR)

(And other underlying mechanisms)

This emphasises the need to research other synthetic lethal relationships

Question 82

Besides PARPi and BRCA1/2, how can we discover more synthetic lethal relationships, and which examples have we discovered so far?

Answer 82

Large scale lethality screens using CRISPR/Cas9 can reveal which genes are synthetically lethal with genes known to be frequently mutated in cancer

It is also important to have a fundamental understanding of how DNA repair pathways interact with each other in both healthy and cancer settings -> understand the underlying mechanisms

1. ATM DEFICIENCY
   - ATM is a major DDR kinase
   - Deficiency is common in many sporadic cancers
   - It is synthetically lethal with inhibitors of several DNA repair enzymes, including:
   -> PARPi’s
   -> TOP1i’s
   -> ATRi’s
   -> POLQi’s
   -> DNA-PKcs inhibitors
2. MICROSATELLITE INSTABILITY (MSI)
   - Mutations in MLH1, MSH2, MSH3 and MSH6 are common in uterine, colorectal, stomach cancer, etc.
   - MSI is synthetically lethal with Werner inhibitors (WRNi’s)