MYC and p53 lectures

Question 1

why is MYC a key oncogene

Answer 1

-it is overexpressed in wide range of cancers such as cervical, breast, colon, lung, melanomas etc, MYC activity found in 90% of human tumour cell samples
-MYC accounts for 15-20% of all genes regulated (1000s)
-MYC gene amplification is frequently detected in 15% of cancers, protein overexpression is seen in 40% of breast cancer, and MYC translocation is seen in other cancers such as Burkitt’s lymphoma
-MYC protein is subject to modifications (PTM) such as phosphorylation, acetylation, and ubiquitination which affects its interactions with other proteins and transcription activity

Question 2

what does MYC act as

Answer 2

mainly as a transcription factor but also partners with other members of its own family for pro-growth and pro-survival functions

Question 3

what is Burkitt’s lymphoma and details of how it occurs in relation to MYC

Answer 3

it is an aggressive B cell malignancy associated with EBV infection
it occurs as a result of c-myc translocating to Ig heavy chain locus which places MYC under highly active transcriptional regulators (note - c-myc translocation alone is not sufficient to cause malignancies)

Question 4

overview of MYC

Answer 4

c-myc protein, short half-life, subject to post translational modifications
Belongs to large family of bHLH transcription factors, full activity needs to dimerise with partner MAX at LZ region and phosphorylation in TAD, translocates to nucleus and binds E-box motif which are found in the promoters of target genes

Question 5

what are some transcriptional targets of MYC/MAX

Answer 5

E2F1, E2F2, E2F3 (transcription factors), and cyclin D2, CDK4, and CUL1

Question 6

how does transcription of MYC/MAX’s targets occur

Answer 6

MYC/MAX heterodimer binds to E-box sequence CACGTG of target genes to regulate gene transcription, multiple coactivator complexes such as TRAPP are involved, TRAPP is part of a complex containing HAT activity

Question 7

what pathways positively regulate MYC expression/stability

Answer 7

Ras, Wnt, Notch, and EGFR

Question 8

what pathways inhibit MYC gene expression/activity

Answer 8

TGFb (expression) and BRCA1 (activity) (these are frequently mutated in cancers)

Question 9

what effect do growth factors/mitogenic signals have on MYC

Answer 9

cause increased levels of MYC

Question 10

what targets MYC for degradation

Answer 10

phosphorylation at Thr58, mutations at or near this residue are common in cancers

Question 11

what is GSK3

Answer 11

a serine/threonine kinase, ubiquitously expressed and in resting cells is constitutively active and is inactivated through phosphorylation by mitogenic signals such as kinase AKT

Question 12

how is MYC stabilised

Answer 12

with phosphorylation at Ser62 by MAPK which is a Ras downstream effector

Question 13

how does MYC promote cell cycle progression

Answer 13

-MYC/MAX regulates CyclinD2 and CDK4 expression
-cyclinD/CDK4 sequesters p27
-MYC is involved in p27 degradation
-MYC transcriptionally regulates CUL1
-CAK then phosphorylates CDK2 leading to active CyclinE/CDK2 which is required for Rb hyperphosphorylation and E2F release
-MYC is able to induce expression genes encoding E2F1, 2, and 3 transcription factors

Question 14

what happens when MYC binds to MIZ1 (TF)

Answer 14

leads to reduced p15 and p21 expression

Question 15

how and why do cancer cells use altered metabolic program

Answer 15

increase glucose uptake and process it mainly by aerobic glycolysis (warburg effect), this provides substrates and energy for tumour growth and accumulates key intermediates (nucleotides, amino acids, and lipids) needed to support growth and cell division

Question 16

how is MYC involved in altered metabolic program

Answer 16

because MYC regulates lactate dehydrogenase A, glucose transporter, hexokinase 2, phosphofructokinase, and enolase 1, and MYC is involved in Ribosome biogenesis

Question 17

how is MYC involved in hTERT expression

Answer 17

MYC activates telomerase by inducing expression of its catalytic subunit TERT
in normal cells telomerase activity is absent and telomerase shortening is thought to act as a signal that a cell is progressing towards the end of its replicative life, however in cancer cells (and STEM cells) telomerase is expressed which serves to lengthen telomerase by adding the repeat sequence TTAGGG

Question 18

how does MYC positively regulates angiogenesis

Answer 18

by upregulating VEGF expression

Question 19

what happens when MYC downregulates E-cadherin expression

Answer 19

leads to increased cell motility invasiveness

Question 20

most cells that acquire a MYC oncogene are rapidly eliminated through apoptosis, how is this evaded in cancer cells

Answer 20

the apoptotic program needs to be deregulated before the mitogenic actions of MYC become apparent

Question 21

what are the mouse and human ARFs

Answer 21

p19ARF is mouse, p14ARF is human, both target p53

Question 22

how is MYC involved in oncogenic stress/ARF expression

Answer 22

MYC leads to increase in E2F activity which increases expression of ARF which then activates p53 by binding to it and sequestering its negative regulator MDM2 thus p53 levels increase in the cell leading to G1 and G2 arrest

Question 23

what happens to ARF and p53 in cancers

Answer 23

ARF locus is often targeted by deletion or its expression is silenced through methylation thus p53 is frequently inactivated in cancers

Question 24

overview of p53

Answer 24

-a nuclear protein (not an oncogene), that normally exists in the cell as a homotetramer
-most commonly mutated protein in cancers (half of all cancer types are from p53 mutation)
-mutant p53 acts in a dominant way to ‘block’ the activity of WT p53, even if the tetramer has a single mutation it will affect the function overall
-p53 is defective in many tumours such as Li Fraumeni syndrome which is increased susceptibility to many types of cancer

Question 25

what do most mutations affecting p53 cause and how are they carried out

Answer 25

-p53 protein to lose its transcription activating powers, carried out through amino acid substitutions in the DNA-binding domain of p53
-also leads to deregulation of apoptotic pathways and increases tumorigenesis

Question 26

how is p53 expression and activity controlled

Answer 26

1-gene transcription where stimuli such as DNA damage or irradiation increase p53 transcription
2-protein stability where p53 is unstable and rapidly degraded in normal cells and p53 levels in the cell increase in response to DNA damage
3-post translational modification where phosphorylation at key serine residues is required for full activity
4-location where p53 needs to be in the nucleus for full activity
5-structural interactions/oligomerization where p53 needs to form a tetramer (4 identical polypeptide subunits) for full activity
6-p53 protein levels increase following phosphorylation events mediated by several kinases which become activated following DNA damage

Question 27

during sensing DNA damage what activates the checkpoint kinases that phosphorylate p53

Answer 27

ATM kinase and ATR kinase

Question 28

how does p53 act as a transcriptional activator

Answer 28

p53 is a transcription factor that positively regulates transcription of many targets as well as halting cell cycle progression in response to DNA damage and attempts to aid in the repair process

Question 29

what is p53 a transcriptional activator of

Answer 29

-p21(CDKI)
-GADD45 (role in repairing DNA damage)
-BAX, Bid, APAF1 (role in apoptosis)
-Thrombospondin1 (anti-angiogenesis)
-MDM2 (p53s negative regulator)

Question 30

how does p53 halt cell cycle progression

Answer 30

if DNA damage is detected in G1 then p53 halts cell cycle progression by transcriptional upregulation of CDKI p21 (increase in p21) then the DNA repair machinery is activated

Question 31

how does an increase in p21 induce DNA repair

Answer 31

p21 causes a halt in the cell cycle where PCNA can then form a complex with DNA polymerase such as Pol delta to facilitate efficient DNA synthesis

Question 32

what are the targets of p53 and what they do

Answer 32

-cyclinB CDK1
-14-3-3delta protein which sequesters cycinB CDK1 complex in the cytoplasm which prevents it from moving into the nucleus thus preventing mitosis until DNA has been successfully repaired
-GADD45 which binds to and dissociates CDK1-cyclinB kinase
-p21 CDKI

Question 33

how does p53 work in the extrinsic apoptosis pathway

Answer 33

-p53 increases transcription of FAS
-p53 enhances FAS transport to cell surface

Question 34

how does p53 work in the intrinsic apoptosis pathway

Answer 34

-p53 induces BAX, PUMA, and NOXA which are pro-apoptotic proteins
-p53 induces Apaf1
-p53 inhibits BCL2 and BCL2XL through direct binding, these are anti-apoptotic/pro-survival proteins that we want blocked in cancer

Question 35

what are other events that can disable p53

Answer 35

nucleotide depletion, hypoxia, exposure to NO, UV, loss of upstream activators such as ATM and Chk2, ionizing radiation, many chemotherapeutic drugs

Question 36

where did the name PRIMA1/PRIMA1met (now known as APR246) come from and what is its affect on p53

Answer 36

p53 Reactivation and Induction of Massive Apoptosis
it (APR246) binds and refolds p53 into its WT conformation thus restoring function to p53

Question 37

what did research into APR246 used as a therapeutic target to triple negative breast cancer show

Answer 37

-APR246 anti-proliferative activity was more potent in mutant p53 vs WT p53 triple negative breast cancer cells
-APR246 induced cell death through apoptosis in a mutant p53 dependent manner
-APR246 inhibited cell migration in p53 mutant cell (implication for metastatic breast cancer)
-p53 protein levels may be a therapeutic predictive biomarker for response to APR246

Question 38

what is the current clinical status of APR246

Answer 38

completes phase 1b trials and ongoing in phase 2 of trials