Chapter 6: Growth inhibition and tumor suppressor genes (book)

Question 1

Are phosphatases often oncogenes or tumor suppressor genes?

Answer 1

Suppressor genes!

Question 2

Can you name some examples of tumor suppressor genes and their corresponding cancer? (DON’T learn this by heart)

Answer 2

RB1 (retinoblastoma, osteosarcoma), WT1 (nephroblastoma), p53 (sarcomas, breast/brain tumors), NF1 (neurofibromas, sarcomas, gliomas), NF2 (schwannomas meningiomas), VHL (hemangioma, renal, pheochromocytoma), APC (colon cancer), INK4a (melanoma, pancreatic), PTC (basal cell carcinoma, medulloblastoma) BRCA1/2 (breast/ovarian tumors) and maaaannnnyyy more

(see table 6.1 if interested, but you don’t need to know this)

Question 3

One gene encoding a phosphatase that is frequently mutated in many cancers is PTEN that has a dual specificity- it can act as both a protein and lipid phosphatase (in the PI3 kinase pathway). What does a mutation mean for this gene?

Answer 3

Loss of the inhibitory dephosphorylation activity of PTEN in the PTEN mutant phenotype may result in constitutively active PI3 kinase pathway, involving activation of protein kinases Akt and m-TOR. The net result is the inhibition of apoptosis and induction of cell proliferation that favors oncogenesis (I think you don’t actually have to know the enzymes, just understand the process and see this as an example)

Question 4

Most phosphatases are tumor suppressors, but what is an exception to this rule?

Answer 4

Ataxia telangiectasia mutated (ATM) kinase (functions in DNA repair) Also PTPN1 can act as both a suppressor as an oncogene

Question 5

Retinoblastoma can be familial or sporadic. What percentage of patients is familial, and what percentage is sporadic?

Answer 5

40% is familial, 60% is sporadic

Question 6

In familial retinoblastoma, all cells already have one germline mutation. What does the second mutation often result from?

Answer 6

Somatic mitotic recombination, during which the normal gene copy is replaced with a mutant copy

Question 7

In which part of the cell cycle is RB active?

Answer 7

In inhibiting the G1 to S-phase transition

Question 8

What are most often the mutation identified that result in the abrogation in RB function?

Answer 8

Deletions, frameshift or nonsense mutations

Question 9

The Rb gene is expressed in all adult tissues, but is only seen in a couple type of cancers when RB is lost. What are these?

Answer 9

Retinoblastoma (eye), small-cell lung carcinomas and osteosarcomas are initiated by loss of Rb

Question 10

p53 plays a role in antioxidant activity. Explain how

Answer 10

Because of ROS, approximately 20,000 bases of DNA per day can be altered in a single cell. p53 upregulates anti-oxidant functions such as glutathione peroxidase 1 and setrins to protect against ROS. This helps prevent cancer

Question 11

What are examples of upstream stress activators and downstream cellular effects (in regard to activating p53)?

Answer 11

Upstream stress activators (that nurture tumor initiation):

• radiation-, dug- or carcinogen-induced DNA Damage
• oncogenic activation
• hypoxia
• low ribonucleotide pools

Downstream cellular effects (respons to stress signals):

• transient or permanent cell cycle arrest
• DNA repair
• apoptosis
• inhibition of angiogenesis

Question 12

What four distinct domains does the p53 transcription factor contain?

Answer 12

• Amino-terminal transactivation domain
• DNA-binding domain containing a Zn2+ion
• Tetramerization domain
• Carboxy-terminal regulatory domain

Question 13

Fill in: Normally, the level of p53 protein in a cell is high/low

Answer 13

Low

Question 14

Fill in: MDM2 modifies the carboxy-terminal domain of p53, and is thus inhibiting/degrading p53

Answer 14

Degrading (by ubiquitination)

Question 15

When happens when MDM2 binds to the p53 transactivation domain?

Answer 15

It transports p53 into the cytoplasm, away from nuclear DNA (modifying the activity of p53)

Question 16

Can you draw a diagram (in your head if needed) about the regulation of p53 by MDM2?

Answer 16

The feedback loop of p53-MDM2 is shown here. p53 induces the transcription of MDM2, whose product negatively regulates p53. MDM2 adds ubiquitin to p53. Low amounts of p53 lead to a reduction of MDM2 expression

Question 17

Is MDM2 the only regulator of p53?

Answer 17

No

Question 18

The upstream activators of p53 utilize three main independent molecular pathways to signal cellular distress. Explain them.

Answer 18

DNA damages caused by ionizing radiation is signaled by two protein kinases:

• ATM (double-stranded breaks) phosphorylates and activates Chk2. Both ATM and Chk phosphorylate p53, disrupting the binding with MDM2
• ATR and Casein kinase II (cellular stres) phosphorylate p53 to disrupt the binding with MDM2

Activated oncogenes, such as Ras, induce activity of protein p14arf, sequestering MDM2 to the nucleolus of the cells

All three pathways prevent degradation of p53 by MDM2

Question 19

Can you globally draw the four downstream cellular effects of p53 and their corresponding genes? (you don’t need to know these by heart, but you should be able to recognize them)

Answer 19

Question 20

What two functions does the p21 (induced by p53) do?

Answer 20

It inhibits several cyclin-cdk complexes, causing a pauze in the G1-S cell cycle. p21 also binds and inhibits PCNA (proliferating cell nuclear antigen)

Question 21

What important proteins are induced by p53 that play a role in the two apoptotic pathways?

Answer 21

Pro-apoptotic (activated): NOXA, PUMA, p53AIP1 and Bax (cause release of cytochrome c and activate apoptosome) Anti-apoptotic (repressed): Bcl-2

Question 22

Which gene is involved in the nucleotide excision repair and is regulated by p53?

Answer 22

XPC

Question 23

What inhibitor of angiogenesis is also regulated by p35?

Answer 23

Thrombospondin

Question 24

What are important factors (GLOBALLY) that affect the downstream effect of p53?

Answer 24

target gene selectivity, level of p53 protein, subcellular localization, co-factors

Question 25

Explain what is happening in this figure

Answer 25

This is an example of molecular factors that mediate the downstream effects of p53: cell cycle arrest or apoptosis? The regulation of a gene that codes for a cyclin-cdk inhibitor, the p21 gene, plays a pivotal role.

• Top: without competition from Myc, p53 and MIZ-1 bind to the promoter of p21 and induce transcription, resulting in cell cycle inhibition.
• Bottom: upon oncogenic activation, Myc competes with the binding of p53. Myc and Mia-1 bind tot he p21 promoter, inhibit transcription and block cell cycle inhibition. ASPP binds to p53 and facilitates activation of apoptotic genes to induce apoptosis

Question 26

Fill in: Over …% of all p53 mutations are missense mutations and result in single amino acid substitutions. More than …% of the missense mutations are located in the DNA-binding domain, and more than …% of these affect only six codons and are therefore referred to as ‘hotspots’ You don’t actually have to learn these numbers, just for indication

Answer 26

Over 75% of all p53 mutations are missense mutations and result in single amino acid substitutions. More than 90% of the missense mutations are located in the DNA-binding domain, and more than 30% of these affect only six codons and are therefore referred to as ‘hotspots’

Question 27

What is the Li-Fraumeni syndrome?

Answer 27

This is a syndrome characterized by a germline, autosomal dominant mutation in the p53 gene. Cancers that are seen in these patients are sarcomas, breast cancer, leukemia and brain tumors. Cancer develops at an earlier age over several generations

Interesting fact: Fraumeni syndrome patients do not always exhibit loss of the wild-type p53 allele, suggesting that haploinsufficiency of p53 is sufficient for tumor formation

Question 28

What are different therapeutic strategies that target the p53 pathway and how they work (globally)

Answer 28

These have been thorouhgly discussed in the lecture, so I will not be explaining them again