L7 - Txnal Regulation of oncogenes and tumour suppressors

Question 1

What does inheritance of one inactive RB allele give?

Answer 1

90% chance of developing retinoblastoma at any early age after somatic mutation of second allele

Question 2

What is mutation of RB gene also common in?

Answer 2

Many tumour types
Almost all small cell lung cancers
In cervical cancers RB neutralised by E7 from HPV that binds to the pocket of RB

Question 3

What is the structure of the RB pocket?

Answer 3

Two subdomains each resembling a cyclin fold that interact to form a single domain

Question 4

What is the structure of the rest of RB?

Answer 4

N terminal domain has cyclin folds
Rest unstructured and flexible
C terminal bound by protein phosphatase 1 and CDK 2,4,6

Question 5

What happens to RB in G1 phase?

Answer 5

Hypophosphorylated by cdk4 and cdk6 +cyclin D committing cell to leave G1 (restriction point)

Question 6

What is the function of cyclin D in the cell cycle/

Answer 6

Expressed in response to mitogens - RB therefore couples cell cycle entry to mitogenic signals (gatekeeper)

Question 7

What happens to RB in S phase?

Answer 7

cdk2/cyclin E phosphorylate further and inactivate RB

Question 8

What happens to RB in G2?

Answer 8

cdk2/cyclin A maintains phosphorylation and inactivation

Question 9

What happens to RB in M phase?

Answer 9

Phosphorylation reversed by protein phosphatase 1 (PP1)

Question 10

What happens when RB is hypophosphorylated?

Answer 10

cells remain in G1 or withdraw into a quiescent state

Question 11

How can some cancers subvert Rb?

Answer 11

Neutralise by binding of oncogenic protein to pocket domain or switched off by phosphorylation

Question 12

How can cancers hypophosphorylate RB?

Answer 12

Overespress Cyclin D and ckd4

Question 13

What is the function of INK4?

Answer 13

Encodes p16 and p15 that bind to cdk4 and 6 blocking action so cant bind to cyclin D

Question 14

How is INK4 involved in cancer?

Answer 14

When INK4 genes deleted (melanoma) this deregulates RB phosphorylation

Question 15

What TFs does RB regulate?

Answer 15

MyoD, UBF, TFIIIB, E2Fs

Question 16

What are E2Fs involved in

Answer 16

Regulate DNA syn genes and cyclin E gene

Recruits TFIID to promoters

Question 17

How does RB regulate E2Fs?

Answer 17

Binds txn action domain
RB recriots HDACs and chromatin remodellers to repress E2F
Phosphor of RB causes release

Question 18

What do activator E2Fs do?

Answer 18

Txn activators during cell cycle progression and most abundant in proliferating cells

Question 19

What do repressor E2Fs do?

Answer 19

Expressed in quiescent cells, inhibit E2F target genes

Question 20

How is RB involved in pol 3 txn?

Answer 20

Hypophos RB binds to TFIIIB and blocks interactions with C + pol 3
So RB can block all pol 3 txn

Question 21

What other proteins have pocket domains?

Answer 21

p107 and p130 (54% identical to each other and 25% identical to RB)

Question 22

What is a characteristic of p107 and p130 and how is this involved in cancers?

Answer 22

Can be switched off through phosphor

So a lot of cancers lose function of all 3

Question 23

What is ARF>

Answer 23

E2F target

One of 3 tumour sup genes in a 35kb region often deleted in tumours

Question 24

How does ARF work?

Answer 24

Induced by oncogenic signals
Binds MDM2 and inhibits ubiquitin ligase activity
Triggers p53 response

Question 25

What does pol 1 do?

Answer 25

Syntehsise 18S, 5.8S and 28S rRNA as large precursor transcript

Question 26

How does ARF affect the activity of pol 1?

Answer 26

Inhibits processing of S's rRNA

Question 27

What are free ribosomal proteins and example of one?

Answer 27

Released by imbalances in ribosome biogenesis (nucleolar stress) eg RPL11

Question 28

What can RPL11 do?

Answer 28

Bind and inhibit MDM2 - activating p53

Question 29

What does activating p53 do during nucleolar stress?

Answer 29

Provides important checkpoint to prevent cell cycle progression when ribosome production is inaequate

Question 30

What are p53super mice and what did they show?

Answer 30

Extra copy of p53 inserted in | Reduced cancer incidence

Question 31

What does loss of RB function cause in cancers?

Answer 31

Induction of ARF and hence p53 leading to oncogenic stress

Question 32

How does HPV cause cancer?

Answer 32

Encodes oncogenincE7 that binds and neutralises and E6 targets p53 for ubiquitination and degradation - causing cervical cancer

Question 33

How does SV40 DNA tumour virus cause cancer?

Answer 33

Large T antigen binds and neutralises RB and p53

Question 34

What is the incidence of loss of p53 in all cancers?

Answer 34

Over 50% (highest of all known genes)

Question 35

Where is p53 usually mutated?

Answer 35

DNA binding domain

Question 36

What do MDM2 and MDMX do?

Answer 36

Bind txn domain of p53 (mono export, poly degrade) | Both bind and inhibit RB

Question 37

What do intentional mutations of p53 in mice show?

Answer 37

Knock in - prevents induction of apoptosis but not tumour suppression p53 mutants able to induce cell cycle arrest and apoptosis, but not supress tumour

Question 38

What occurs in Brukitt lymphoma?

Answer 38

Chromosomal translocations place MYC under control of an Ig enhancer

Question 39

How does MYC generally act?

Answer 39

Stimulates txn of open genes rather than inducing silent ones

Question 40

How does the way MYC usually acts cause cancer?

Answer 40

May amplify other oncogenic TFs | contrasts normal assumption that oncogenic transformation involves switching key genes or or off

Question 41

How did they show what genes MYC activated and repressed?

Answer 41

Model cell line carrying MYC transgene controlled by a doxycycline-activated promoter

Question 42

What genes did they show MYC activated and repressed?

Answer 42

Increased pol2 occupancy at KLF15 | Reduced pol 2 occupancy at ALDJ3B1

Question 43

How can repression by MYC sometimes work and give an example?

Answer 43

Involving interference with other activators | Miz1 activates p21, but MYC blocks interaction with p300

Question 44

How is MYC involved with pol1?

Answer 44

DNA sites recognised by MYC found in rRNA promoters, binds and stimulates txn but recruiting SL1 and HATS

Question 45

How is MYC involved with pol3 txn?

Answer 45

MYC recruited by TFIIIB where it then recruits HATs to stimulate txn

Question 46

What does the action of MYC have to do with cancer?

Answer 46

Involved in all protein synthesis required for tumour growth

Question 47

How did they show MYC induced increased protein synthesis?

Answer 47

Used MYC overexpresser in mice B cells, stimulated protein syn and size of b cells Then crossed with a lower translating mutant (L24) and reverted to normal (control)

Question 48

What was a therapeutic option for MYC driven cancers?

Answer 48

L24 decreased lymphomagenesis suggesting MYC needs to increase rate of protein syn to cause lymphomas