Cancer

Question 1

What is the definition of cancer?

Answer 1

heterogeneous group of around 200 diseases characterised by:

• uncontrolled cell growth
• invasion and metastasis
• undifferentiated cells
• dysregulation of cell signalling responses

Question 2

What is carcinogenesis?

Answer 2

the process of cancer formation driven by mutations and epigenetic changes in somatic tissues

Question 3

Define hyperplasia and dysplasia?

Answer 3

Hyperplasia = increased cell division

dysplasia = cells change form

Question 4

What is the difference between a benign and malignant neoplasm?

Answer 4

Benign neoplasm = in situ localised cell overgrowth

malignant neoplasm = invasion of surrounding tissue and ability to metastasise

Question 5

What are the names of the cancers derived from the following tissues?

1) Epithelium
2) Stromal tissue
3) Blood

Answer 5

1) carcinoma (90% of all human cancers)
2) sarcoma
3) leukaemia, lymphoma

Question 6

Why are actively dividing cells predisposed to forming tumours?

Answer 6

rapid cell division so increase in probability of unrepaired DNA replication errors so increase in probability of mutations

Question 7

Are are tumour cells different?

Answer 7

Yes but they are monoclonal (meaning they derived from the same starting cell - but have picked up mutations along the way so are not identical)

Question 8

Describe how mutation drives the formation of cancer

Answer 8

1) mutation in single cell leading to reduced control of cell division and clonal expansion producing a benign tumour
2) rapid division increases rate of other mutations and results in development of another mutations that confer growth advantage and result in clonal expansion, rapid division, and escape of checkpoints to form a malignant tumour
3) malignant tumour invades surrounding tissue and cancer cells can enter the bloodstream or lymphatics where they are transported to secondary sites within the body

Question 9

What is the difference between a driver mutation and a passenger mutation?

Answer 9

A driver mutation directly or indirectly confers a selective growth advantage to the cell allowing for increased proliferation and evasion of checkpoints

A passenger mutation doesn’t confer a selective growth advantage in any way, but may affect morphology or metabolic pathways

Question 10

What are the three positive and three negative regulators of cell proliferation?

Answer 10

Positive regulators:

1) classical oncogenes (stimulate proliferation)
2) telomerase (stimulates proliferation)
3) anti-apoptotic genes (inhibit apoptosis)

Negative regulators:

1) classical tumour suppressor genes (inhibit proliferation)
2) indirectly acting tumour suppressor genes (E.g. in genome maintenance) (inhibit proliferation)
3) apoptotic genes (stimulate apoptosis)

Question 11

How is the balance between cell proliferation and apoptosis regulated?

Answer 11

endogenous and exogenous signals activate signalling pathways that may stimulate proliferation (E.g. mitogen signalling activates transcription factors that express proliferative genes) or arrest cell cycle (E.g. growth inhibiting factor signalling activates transcription factor that results in expression of cell cycle inhibitors)

Question 12

List the events of the cell cycle including the checkpoints

Answer 12

G1 phase = growth before chromosome replication
(G0 = quiescent cells)
RESTRICTION POINT CHECKPOINT (favourable environment - if not = remain in G0)
S phase = DNA synthesis produces two identical sister chromatids
S-PHASE CHECKPOINT
G2 phase = growth and preparation for mitosis, mitotic spindle begins to form
G2/M CHECKPOINT (all DNA replicated, favourable environment - if not - remain in G2)
M phase = mitosis and cytokinesis
METAPHASE TO ANAPHASE CHECKPOINT (all chromosomes attached to spindle correctly)

Question 13

What proteins drive the progression through the cell cycle?

Answer 13

cyclin dependent kinases (Ser/Thr kinase activity only present when bound to its cyclin)

Question 14

How do cyclin and CDK levels vary throughout the cell cycle?

Answer 14

Cdk proteins are constitutively expressed throughout the cell cycle

Cyclin expression oscillates (rise and fall) throughout the cell cycle so they are only expressed when the Cdk activity is required.

Question 15

What are the cyclin/CDKs present at each stage of the cell cycle?

Answer 15

G1-Cdk = cyclin D - Cdk 4/6

G1/S-Cdk = cyclin E - Cdk2

S-Cdk = cyclin A-Cdk 2 (SPF)

M-Cdk = cyclin B - Cdk 1 (MPF)

Question 16

How do cyclin-Cdk complexes drive cell cycle progression?

Answer 16

• activate the next cyclin-Cdk complex (E.g. by promoting expression of the next cyclin)
• proteolysis of key enzymes

Question 17

How is M-Cdk activity regulated?

Answer 17

1) association with cyclins
2) activation by phosphorylation of Thr160 by Cdk-activating kinases (CAKs)

3) phosphorylation status of Try15 and Thr14 of M-Cdk
- Wee1 kinase phosphorylates Try15 and Thr14 = inhibit
- Cdc25 phosphatase dephosphorylates Tyr15 and Thr14 = activates

4) CDK inhibitor proteins (CDKIs) inhibit Cdks

Question 18

How does G1-Cdk drove progression through the restriction point checkpoint?

Answer 18

in G0 and early G1 = transcription factor E2F is bound to, and inhibited by, Rb protein.

Mitogens bind to receptor, results in conformational change that results in phosphorylation of the cytoplasmic domain of the receptor. Activation of signalling pathway that results in activation of Ras and leads to a MAPK signalling cascade that ends in transcriptional response:
- activation of Myc transcription factor leads to cyclin D expression

Cyclin D then associates with Cdk4/6 to form the G1-Cdk.

• G1-Cdk phosphorylates the Rb protein, causing E2F to be released.
• E2F activates transcription of genes involved in the G1/S transition (E.g. cyclin E, cyclin A, enzymes for DNA synthesis)

Question 19

What type of gene is Rb?

Answer 19

tumour suppressor (Rb protein binds to E2F and prevent cell cycle progression until sufficient growth factor signalling)

Question 20

How does the M-Cdk/MPF promote the G2/M transition?

Answer 20

• induces spindle assembly
• initiate chromosome condensation
• ensures sister chromatids are attached to opposite spindle poles
• promotes breakdown of nuclear envelope
• rearranges the actin cytoskeleon
• rearranges the Golgi apparatus

Question 21

How is the regulation of the G2/M transition regulated?

Answer 21

Phosphorylation status of M-Cdk/MPF:

• Wee1 kinase phosphorylates Y15 and T14 to inhibit activity of MPF
• expression of Cdc25 phosphatase in late G2/M phase results in removal of inhibitory phosphates and activation of MPF

Question 22

How do cyclin-Cdks drive cell cycle forward by proteolysis of key proteins?

Answer 22

• Phosphorylate cell cycle regulators = substrates for the SCF ubiquitin ligase complex
• phosphorylate ubiquitin ligases to activate them (E.g. APC - anaphase promoting complex)

Question 23

What two things does progression through the cell cycle depend on?

Answer 23

1) activation of specific Cdk activity (specific to phase)

2) elimination of proteins from previous cell cycle phases by ubiquitin mediated proteolytic degradation

Question 24

Describe the action of SCF ubiquitin ligase complex controls the G1/S phase transition?

Answer 24

has three subunits: Skp1, Cullin, and F-box protein

• p27 Cdk inhibitor (CDKI) inactivates S-Cdk/SPF during G1
• phosphorylation of p27 by the G1/S-Cdk to target it for polyubiquitination by SCF and subsequent degradation in the proteasome
• S-phase SCF ubiquitinates G1 factors (involved in replication origin licensing) that prevent entry into S phase, and targets them for proteasomal degradation
• Cyclin E (of G1/S-Cdk) is targeted for degradation by SCF aswell

Question 25

How is M-Cdk/MPF activity regulated by the APC ubiquitin ligase?

Answer 25

Cdc20 expression in G2 phase binds to APC, activating it, promoted by phosphorylation by M-Cdk - results in ubiquitination of cyclin B of MPF by APC and subsequent degradation by the proteasome.

Question 26

How does the APC-Cdc20 ubiquitin ligase promote the metaphase to anaphase transition?

Answer 26

- M-Cdk/MPF phosphorylates APC to allow binding to Cdc20 - APC-Cdc20 degrades securin, which activates separase - Separase cleaves cohesin complex holding the two sister chromatids together, promoting anaphase

Question 27

How does APC-Cdh1 ubiquitin ligase ensure exit from mitosis and control of G1? ********************************************************************

Answer 27

Exit from M phase: - Degradation of M-Cdk cyclin B by APC-Cdc20 during M phase results in reduced phosphorylation of APC so dissociation between APC and Cdc20 - APC binds alternative activator Cdh1, which continues to ubiquitinate M-Cdk cyclin B for degradation allowing exit from M phase Control of G1 phase: - APC-Cdh1 remains active in early G1 - Increase in G1/S-Cdk (Cyclin E - Cdk2) phosphorylates and inactivates Cdh1 - APC remains inactive until activated by association with Cdc20 in M phase.

Question 28

What are responses when errors are detected by cell cycle checkpoints?

Answer 28

1) activation of DNA repair 2) cell cycle arrest 3) inhibit further DNA synthesis (S-phase checkpoint)

Question 29

What is the structure of a checkpoint pathway (E.g. for a DNA double-stranded break)

Answer 29

- damage specific sensors bind to the damaged DNA (MRN and KU for DSB) - sensors activate transducer regulatory kinases to initiate the damage response (ATM for MRN and DNA-PKCS for KU) - transducers activate effectors, which perform checkpoint functions (E.g. DNA repair proteins, proteins that arrest cell cycle) - prolonged arrest leads to apoptosis in multicellular organisms

Question 30

Describe the activation of the spindle checkpoint (metaphase to anaphase checkpoint) **********************Cdc20??******************************

Answer 30

- lack of attachment, incorrect attachment or chromosomes to spindle - monitored by spindle assembly checkpoint - APC-Cdc20 is the main effector and is activated to prevent transition into anaphase and mitotic exit

Question 31

How do mitogens promote cell division/passage through restriction point in normal cells?

Answer 31

- Mitogen signalling activates TOR kinase signalling pathway that results in increase in protein synthesis to increase the size of a cell. - mitogens stimulate cell division by activating cyclin-Cdks (mitogen signalling results in MAPK phosphorylation cascade that activates transcription factors that increase expression of G1 and S phase cyclins) - E.g. Myc transcription factor increase expression of cyclin D (G1 cyclin)

Question 32

What is an oncogene? what happens when mutated?

Answer 32

Stimulates cell proliferation or inhibit apoptosis as a proto-oncogene when mutated, this leads oncogenes that increase the activity of a protein

Question 33

What are the 10 hallmarks of cancer?

Answer 33

1. Self-sufficiency in growth signalling 2. ability to evade growth suppressors 3. Activating invasion and metastasis 4. Enabling replicative immortality 5. Inducing angiogenesis 6. resistance to apoptosis 7. de-regulating cellular energetics 8. Avoiding immune destruction 9. Genome instability and mutation 10. Tumour promoting inflammation

Question 34

Oncogenic mutation are genetically ___________

Answer 34

dominant (mutation of a single allele is sufficient to contribute to carcinogenesis)

Question 35

What are the six major classes of cellular oncogenes?

Answer 35

1. mitogens 2. mitogen receptors 3. signal transduction component 4. transcription factors 5. cell cycle drivers 6. cell death inhibitors.

Question 36

What are the three types of oncogenic mutations (turn a proto-oncogene into an oncogene) and what are their results?

Answer 36

1) Translocation (gene moved to new locus so now under control of a poorly controlled or constitutively active promoter) = excess protein 2) Gene amplification (multiple copies of the gene) = excess protein 3) Point mutation (more common) = hyperactive or degradation-resistant protein

Question 37

Give an example of a mitogen receptor oncogene: | - name, types of mutation, results of mutation

Answer 37

name = EGFR (epidermal growth factor receptor) Types of oncogenic mutations: - amplification = more receptors = increased signalling - deletion = truncated receptor lacking extracellular domain = constitutively active in absence of mitogen Results: - promotes self-sufficiency in growth signalling by constitutive activation of pathway from EGFR

Question 38

Give an example of a signal transduction component oncogene: | - name, types of mutation, results of mutation

Answer 38

name = Ras protein - small GTPase (hydrolyses GTP to GDP to inactivate itself) - mitogen binds to receptor causing GDP exchanged for GTP, activating Ras - Ras activates signalling cascade via Raf kinase Types of oncogenic mutation: - mutations at codons 12, 13, and 61 = prevent GTPase activity = constitutively active Ras Results: - promotes self-sufficiency in growth signalling by constitutive activation of pathway from Ras

Question 39

Give an example of a transcription factor oncogene: | - name, types of mutation, results of mutation

Answer 39

name = Myc protein - stimulates cell growth and division in response to mitogen signalling Types of oncogenic mutation: - gene amplification = increased expression of Myc = hundreds of copies resulting from rearrangements that bring sequences from several chromosomes together - point mutation = stabilises Myc = resistant to degradation - translocation = under control of more active promoter so increased expression Results: - promotes self-sufficiency in growth signalling

Question 40

What is the cause of Burkitt's Lymphoma?

Answer 40

Translocation brings the MYC gene under control of sequences that normally drive the expression of antibodies in B cells - MYC mutant B cells proliferate to form a tumour

Question 41

Give an example of a cell cycle regulator oncogene: | - name, types of mutation, results of mutation

Answer 41

name = Cyclin D - Cdk4 (G1-Cdk) - CCND1 gene encodes cyclin D - CDK4 gene encodes Cdk4 Types of oncogenic mutation: - amplification: - amplification of CCND1 results in over-expression of cyclin D - amplification of CDK4 results in over-expression of Cdk4 = inappropriate expression of G1-Cdk = unregulated phosphorylation of Rb = release of too much E2F = increase transcription of cyclin E and cyclin A = drives progression through restriction point in absence of mitogen signalling - mutation within CDK4 results in Cdk4 resistant to p16 inhibition so promotes inappropriate entry into S phase Results: - promotes self-sufficiency in growth signalling

Question 42

What are the characteristic changes of a cell undergoing apoptosis?

Answer 42

cell shrink and condense cytoskeleton collapses - results in blebbing of plasma membrane nuclear envelope disassembles nuclear chromatin condenses and breaks into fragments cell surface chemically altered - signals for engulfment by neighbouring cell or macrophage

Question 43

What are the two pathways of apoptosis?

Answer 43

Extrinsic pathway - triggered by extracellular signal proteins binding to cell surface death receptors Intrinsic pathway - release of mitochondrial proteins into cytosol that are normally present in the intermembrane space

Question 44

How is the intrinsic pathway of apoptosis regulated by Bcl2 family proteins? and how do mutations in these genes lead to cancerous phenotype?

Answer 44

Anti-apoptotic proto-oncogenes activated to increase the levels of Bcl2 blocks apoptosis and results in immortalised cells that resist cell death. Pro-apoptotic tumour suppressor gene activated to increase the levels of Bax, which forms a Bax-Bax homodimer which induces apoptosis Mutations leading to cancerous phenotype: - oncogenic mutation within anti-apoptotic gene can result in constitutive expression of Bcl2 and result in cancerous phenotype - mutation within the pro-apoptotic gene can switch off the tumour suppressor gene and lead to decreased activation of pro-apoptotic pathways result in cancerous phenotype = both lead to cells resisting apoptosis

Question 45

How does oncogene MDM2 result in cancerous phenotype? | role, types of mutation, results of mutation

Answer 45

Role: A ubiquitin ligase that regulates levels of p53 (a tumour suppressor gene that prevents progression through the cell cycle when DNA is damaged, can promote apoptosis) - no DNA damage = MDM2 ubiquitinates Lys residues in p53 C-terminal domain = degradation, remaining p53 is exported from nucleus - DNA damage = kinase activity (ATM, ATR, CHK2) results in phosphorylation MDM2 and p53 = prevents interaction = allows p53 tetramerises and accumulation Types of oncogenic mutation: - Amplification of MDM2 prevents accumulation of p53 (associates with more p53 and mediate degradation at a higher rate) Results: - Evasion of growth suppressors - resistance to apoptosis

Question 46

How does oncogene telomerase result in cancerous phenotype? | role, types of mutation, results of mutation

Answer 46

Role: - prevents shortening of telomeres (In most somatic cells, telomerase in inactive - so, if telomere length becomes critical, cells withdraw from the cell cycle) Types of oncogenic mutation: - mutation results in overexpression = reactivates telomerase to prevent telomere shortening and allow indefinite cell division. Results: - replicative immortality

Question 47

What is a tumour suppressor gene?

Answer 47

normal role of TSG products is inhibition of cell division: - negative regulation of proliferative signalling - inhibition of cell division - pro-apoptosis

Question 48

How can mutations within a tumour suppressor gene lead to cancer?

Answer 48

Mutations resulting in a decrease in the normal activity of the tumour suppressor protein lead to loss of cell division inhibition

Question 49

Mutations in classical tumour suppressor genes are genetically ___________

Answer 49

Recessive (both alleles need to have the mutation to get an increased risk of cancer)

Question 50

What is retinoblastoma?

Answer 50

- retinal cancer arising in immature retinal cells - affects 1 in 20000 children (routine screening in UK) - 87% of children worldwide with retinoblastoma die (almost exclusively in countries lacking good screening) can be sporadic = no family history, unilateral, single tumour in one eye can be hereditary = bilateral, multiple tumours (disease appears dominant)

Question 51

What is Knudson's two hit mutation hypothesis?

Answer 51

Hereditary retinoblastoma - one mutation is inherited, second mutation is sporadic Sporadic retinoblastoma - both mutations are sporadic

Question 52

Mutations in which gene cause retinoblastoma?

Answer 52

RB tumour suppressor gene

Question 53

How can the second hit mutation cause the loss of the second wildtype RB allele?

Answer 53

Can occur at the chromosome level resulting in loss of heterozygosity: - loss of chromosome carrying wildtype allele and replaced by duplication of the chromosome carrying the mutated allele - mitotic recombination between non-sister chromatids (most common) Can occur at the gene level resulting in loss of gene but not loss of heterozygosity: - gene inactivation - epigenetic silencing (chromatin rearrangement that affects transcription)

Question 54

What is the normal role of the RB?

Answer 54

- Binds to E2F and prevents its activation - G1-Cdk activation results in phosphorylation of Rb, releasing E2F, which acts as a transcription factor to increase expression of the genes required for G1/S transition (E.g. cyclin A, cyclin E)

Question 55

What are the consequences in rb/rb cells?

Answer 55

- Rb protein is often truncated causing it to be unstable and unable to bind to E2F - E2F free to activate genes required for G1/S transition and S phase = cell progression and proliferation - results in sustained proliferative signalling

Question 56

What is the most commonly mutated gene in humans cancers?

Answer 56

p53 (approximately 50% of all sporadic cancers)

Question 57

what is p53?

Answer 57

A tumour suppressor gene that encodes for the p53 protein (a transcription factor). Its main function is to arrest the cell cycle to allow time for DNA damage repair

Question 58

How is p53 an effector of DNA damage checkpoints?

Answer 58

p53 acts downstream of effectors and transducers of DNA damage pathways: - ATM, ATR, CHK2 = phosphorylate Ser to activate N-terminal - MDM2 = ubiquitination of C-terminal Lys - p300 = acetylation of C-terminal Lys

Question 59

How does p53 accumulate in response to DNA damage?

Answer 59

- no DNA damage = MDM2 interacts with p53 and ubiquitinates Lys residues in p53 C-terminal domain = degradation, remaining p53 is exported from nucleus - DNA damage = kinase activity (ATM, ATR, CHK2) results in phosphorylation MDM2 and p53 = prevents interaction = allows p53 tetramerises and accumulation

Question 60

What are the results of p53 accumulation?

Answer 60

Interacts with proteins: - such as p300 (which acetylates histones to enhance transcription and acetylates p53 C-terminal Lys) Promote transcription of target genes that promote cell cycle arrest and cell death: - p21 tumour suppressor gene, which binds to and inhibits the activity of Cyclin E - Cdk2 (G1/S-Cdk) to prevent progression through the cell cycle - GADD45, which binds to PCNA and blocks function as processivity factor (during S-phase replication checkpoint) - 14-3-3sigma tumour suppressor gene, which sequesters Cdc25 phosphatase required for activation of M-Cdk = arresting cells at G2/M boundary - Bax tumour suppressor gene, which is pro-apoptotic - FAS death receptors on cell surface to promote extrinsic apoptosis pathway - APAF1, which activates caspases involved in the intrinsic apoptosis pathway

Question 61

How do mutations in the p53 gene result in cancerous phenotype?

Answer 61

Mutation results in decreased level of p53: - no activation of DNA damage checkpoint - no cell cycle arrest (DNA repair doesn't occur and cells keep dividing) - balance between Bax and Bcl2 shifts towards Bcl2 anti-apoptotic pathway and cell immortality

Question 62

Failure of checkpoints leads to what?

Answer 62

genetic instability, which can lead to accumulation of different mutations

Question 63

Failure of the spindle (M) checkpoint can lead to what?

Answer 63

Aneuploidy

Question 64

Failure of the chromosome duplication checkpoint can lead to what?

Answer 64

Polyploidy

Question 65

Failure of DNA damage checkpoint can lead to what?

Answer 65

Translocation (can result in oncogenes) Deletion |(loss of TSG, constitutively active oncogene) Amplification (oncogenic if occurs within a proto-oncegene)

Question 66

How can p53 activity be lost via inactivating mutation?

Answer 66

- may be caused by specific environmental exposure: Skin cancers = UV signature mutations C --> T or CC --> TT transition Lung cancers = mutations in DNA bases subject to attack by polycyclic hydrocarbons in tobacco smoke

Question 67

Describe how some p53 mutations have a dominant negative effect

Answer 67

mutation of once copy is sufficient to disrupt the p53 protein function because the mutated subunits still tetramerise and disrupt the function of the entire protein and prevent activity.

Question 68

How can p53 activity be lost via degradation?

Answer 68

Cervical cancer - main cause is HPV types 16 and 18 (account for 70% of cases) - HPV produces oncoproteins E6and E7 - E6 binds to p53, targeting it for degradation and preventing accumulation of p53 in response to DNA damage - E7 sequesters Rb, resulting in constitutively active E2F, which drives the cell through the G1/S checkpoint

Question 69

What are the two main ways that p53 activity can be lost?

Answer 69

- inactivating mutation (skin and lung cancers) | - degradation (cervical cancers)

Question 70

Give four examples of gatekeepter TSGs (directly involved in restraining cell proliferation) - name and consequence of mutation?

Answer 70

p53 = mutation results in loss of G1/S and G2/M checkpoints p21 = mutation results in loss of G1/S and S checkpoints RB = mutation promotes proliferation and activity of E2F Bax = mutation results in failure to promote apoptosis of damaged cells

Question 71

Give two hereditary cancer syndromes that result from loss of gatekeep TSGs - name of syndrome, mutant gene, inheritance, and main types of cancer

Answer 71

syndrome = Retinoblastoma Mutant gene = RB1 Inheritance = dominant (inherit one mutant gene to create high risk of cancer susceptibility) Types of cancer = retinoblastoma and osteosarcoma (later in life) Syndrome = Li-Fraumeni Syndrome Mutant gene = p53 inheritance = dominant Types of cancer = Sarcomas, breast, leukaemias, adrenal, brain

Question 72

What is the difference between a gatekeeper and a caretaker TSG?

Answer 72

Gatekeeper TSG = directly involved in restraining cell proliferation Caretaker TSG = maintains the integrity of the genome

Question 73

Mutations in which type of TSGs are commonly associated with familial cancer (and rarely seen in sporadic cancers)?

Answer 73

Mutations in caretaker TSGs

Question 74

Give three examples of hereditary caretaker mutations that are associated with increased cancer risk?

Answer 74

1. Lynch syndrome - autosomal dominant inheritance of a mutant allele of one of the mismatch repair genes (MSH2, MLH1, PMS1, PMS2) - tumours show LOH 2. Xeroderma pigmentosum - autosomal recessive inheritance of defective nucleotide excision repair capability - cannot repair UV inflected DNA damage 3. Familial breast cancer - dominant inheritance of mutation within BRCA1 or BRCA2 nuclear protein genes (promote repair of DSBs by high-fidelity homology directed repair mediated by Rad51) - -> tumours show LOH (abnormal chromosomes due to DSB repair by NHEJ)