Oncogenesis and Tumour Suppressor Genes

Question 1

What are the Major Functional Changes in Cancer?

Answer 1

“1. Increased Growth:

• Loss of growth regulation.
• Stimulation of environment promoting growth e.g angiogenesis.
  2. Failure to undergo programmed Apoptosis or Senescence.
  3. Loss of Differentiation:
  
  • Including alterations in cell migration and adhesion.
    
    4. Failure to Repair DNA Damage:
• Including Chromosomal Instability. “

Question 2

What do Oncogenes and Tumour Suppressor Genes do?

Answer 2

“Oncogenes:

• Normally components of Growth Factor Signalling Pathways.
• When mutated produce products in higher quantities.
• Or produce altered products have increased activity and therefore act in a dominant manner

Tumour Suppressor Gene:

• Products act as a stop signal to uncontrolled growth.
• They may inhibit the cell cycle or trigger Apoptosis. “

Question 3

What is an Oncogene?

Answer 3

“Altered Gene whose product can act in a Dominant fashion to help make a cell Cancerous:

• It arises from a Mutation in a Proto Oncogene.
• A Proto Oncogene is normally involved in the control of cell growth or division.
• A Single Allele Mutated is enough to alter it.”

Question 4

What changes in an Oncogene that leads to Cancer?

Answer 4

“1. Oncogenes are like an Accelerator Pedal.
2. Their job is to make cells divide.
Driving cell division forward
3. Cancer pick up mutations mean that they are Permanently Active.
Like putting a brick on the accelerator.
4. The car approaches the red light and can’t stop.

Oncogene = Gain of Function”

Question 5

What is an Tumour Suppressor Gene?

Answer 5

“Gene whose normal activity Prevents formation of Cancer:

• Both genes for the Tumour Suppressor must be mutated
• Loss of this function by mutation enhances the likelihood that a cell can become cancerous.
• A normal process to maintain control of cell division is lost.”

Question 6

What changes in a Tumour Suppressor Gene that leads to Cancer?

Answer 6

“1. Tumour Suppressor Genes are like the Brakes
2. Even if there is a mutation in an Oncogene that pushes cell division forward.
If the Tumour Suppressor Genes are strong enough they should still be able to counteract the Oncogene.
3. In Cancer pick up mutations that switch the Gene Off.
This is like cutting the brakes in a car.
4. Even if there is no Oncogenic brick on the accelerator without brakes the car won’t stop.

Tumour Suppressor Gene = Loss of Function”

Question 7

Describe the experiment done to prove Sarcoma was Virus related?

Answer 7

“1. Sarcoma sample from chicken grinded.

2. Passed through a fine pore filter.
3. Inject filtrate into another younger chicken.
4. Found that sarcoma developed in the younger chicken.”

Question 8

How does the Rous experiment prove that Sarcoma is Virus linked?

Answer 8

”- The cycles could be repeated indefinitely..

• The Carcinogenic agent was small enough to pass through a filter.
• Although the filter used excluded bacteria it was not small enough to exclude Viruses.
• A Virus must be responsible for the induction of tumour formation.
• Discovery that this Sarcoma was transmissible through Viruses = Rous Sarcoma Virus.”

Question 9

Describe how the Rous Sarcoma Virus induced an Oncogene?

Answer 9

“Oncogenic transformation by this Virus is caused by an extra gene contained in its genome:

• This is an Oncogene called V-src
• Homologous sequences were found in uninfected chickens and other organisms.
• Some genes of Cancer causing Viruses are mutated forms of the Cellular Gene and NOT Viral Genes.
• Rous Sarcoma Viral Gene was a host gene that had been ‘kidnapped’ by the virus and transformed into an Oncogene.
• During Evolution the Virus can acquire fragments of Genes from the Host at Integration Sites and this results in the creation of Oncogenes. “

Question 10

What is the Normal Gene and what does it Mutate into?

Answer 10

“Oncogenes are altered forms of normal genes which are Proto - Oncogenes.
1. The Normal Gene: C-src: Cellular Proto - Oncogene.
2. The Mutated Gene: V-src: Oncogene.
The altered form is transduced by Retroviruses.”

Question 11

What is produced from the Oncogene and how does it induce Cancer?

Answer 11

“The Oncogene product was characterised as a 60kDa Intracellular Tyrosine Kinase.
It can Phosphorylate cellular Proteins and affect Growth.”

Question 12

How does the swapping of C-src with V-src induce Cancer?

Answer 12

“1. V-src oncogene is responsible for causing Cancer.
2. Hybridisation experiments showed that the C-src gene was present in the genome of many species.
3. The Host Cell C-src gene is normally involved in the positive regulation of cell growth and cell division.
4. Following infection however the V-src Oncogene is expressed at high levels in the Host Cell.
Leading to uncontrolled Host Cell Growth,
Unrestricted Host Cell Division and Cancer.”

Question 13

What factors can transform cells into becoming Cancerous?

Answer 13

”- Radiation.
- Chemical Carcinogens.
- Exogenously added Viruses.
These factors may transform cells by switching on the Endogenous Oncogenic information.
Turning a Proto Oncogene into an Oncogene.”

Question 14

What is Viral Oncogenesis and how does it work?

Answer 14

“Viral Oncogenes can be transmitted either by DNA or RNA Viruses:

• DNA Viruses can cause Lytic Infection leading to the death of the cellular host or can replicate their DNA.
• Along with that of the host and promote Neoplastic Transformation.

15-20% of all Human Cancers are caused by Oncoviruses.”

Question 15

Describe DNA and RNA Viruses?

Answer 15

“DNA Viruses:
- Encode various proteins along with environmental factors can initiate Tumours.

RNA Viruses:

• Integrate DNA copies of their genomes into the genome of the Host Cell.
• And as these contain transforming Oncogenes they induce Cancerous Transformation of the Host.”

Question 16

How widespread are Oncogenes and how are they made?

Answer 16

“Over 100 identified Oncogenes
There are examples of Oncogenes for every type of protein involved in a Growth Factor Signal Transduction Pathway.
Proto - Oncogenes are captured by animal retroviruses and are altered in Human Cancer making Oncogenes.”

Question 17

What type of mutations can lead to over activation and Oncogenes?

Answer 17

”- Insertions.

• Amplifications.
• Duplication.
• Translocations.”

Question 18

What is the main consequence of these mutations?

Answer 18

“This leads to loss of response to Growth Regulatory Factors.
Only one allele needs to be altered.”

Question 19

What 4 Proteins are involved in the Transduction of Growth Signals?

Answer 19

“1. Growth Factors.

2. Growth Factor Receptors.
3. Intracellular Signal Transducers.
4. Nuclear Transcription Factors.”

Question 20

How do most Oncogenes proteins exist?

Answer 20

“The majority of Oncogene Proteins function as elements of the Signaling Pathways,
That Regulate Cell Proliferation and Survival in response to Growth Factor Stimulation.”

Question 21

Give an Example of Oncogene proteins acting as Growth Factors:

Answer 21

“Epidermal Growth Factor (EGF):

• Growth Factor Receptors: ErbB
• Intracellular Signalling Molecules (Ras and Raf)
• Ras and Raf activate the ERK MAP Kinase Pathway.
• Leading to the induction of additional genes e.g (fos)
• That encode potentially Oncogenic transcriptional Regulatory Proteins.”

Question 22

What are RAS proteins and what is their significance in Cancer?

Answer 22

“RAS proteins are small GTPases that are bound to GDP in a neutral state.
- Oncogenic activation of RAS is seen in about 30% of Human Cancer.
- Most commonly mutated Oncogene.
- Point Mutations in codons : 12,13 and 61.
Glycine to Valine: Bladder Carcinoma.
Glycine to Cysteine: Lung Cancer. “

Question 23

Outline the steps of the the normal Ras Cascade:

Answer 23

“1. Binding of Extracellular Growth Factor Signal.

2. Promotes recruitment of RAS proteins to the Receptor Complex.
3. Recruitment promotes RAS to exchange GDP with GTP.
   Thus making Activated RAS.
4. Activated RAS then initiates the remainder of the signalling cascade.
   These are the Mitogen Activated Protein Kinase.
5. These Kinases Phosphorylate targets such as transcription factors to promote expression of genes important for growth and survival.
6. RAS hydrolyses GTP to GDP fairly quickly which turns itself off. “

Question 24

How does a mutation affect the Ras Cascade:

Answer 24

As a consequence of mutations there is loss of GTPase activity of the RAS protein normally required to return active RAS to the inactive RAS.

Question 25

What is the MYC Oncogene family?

Answer 25

“MYC Oncoproteins belong to a family of transcription factors that regulate the transcription of at least 15% of the entire genome.
Originally identified in avian myelocytomatosis virus. “

Question 26

What are the 3 MYC Oncogenes?

Answer 26

“1. C-MYC encoding c-Myc protein.

2. MYCN encoding N-Myc protein.
3. MYCL encoding L-Myc protein.”

Question 27

List the downstream effects of MYC:

Answer 27

”- Ribosome Biogenesis

• Protein Translation
• Cell Cycle Progression and Metabolism
• Orchestrating a broad range of biological functions such as Cell Proliferation
• Differentiation
• Survival
• Immune Surveillance “

Question 28

How is the MYC Oncogene linked to Cancer?

Answer 28

“MYC Oncogene is overexpressed in the majority of human cancers.
Contributes to the cause of at least 40% of tumours.”

Question 29

How does MYC activate Gene Expression?

Answer 29

“It encodes a Helix-Loop-Helix Leucine Zipper Transcription Factor.
That dimerises with its partner protein Max to transactivate gene expression.”

Question 30

How are MYC Oncoproteins activated?

Answer 30

“Generally MYC is activated when it comes under the control of foreign Transcriptional Promoters.
This leads to a deregulation of the Oncogene that drives relentless proliferation.
Such activation is a result of Chromosomal Translocation.”

Question 31

What is Burkitt’s Lymphoma?

Answer 31

“Epstein Barr Virus is associated with Burkitt’s Lymphoma.
BL is a high grade lymphoma that can affect children from the age of 2-16

Classical African/Endemic BL:
- In central africa children with chronic malaria infections have a reduced resistance to the virus. “

Question 32

Describe the genetic changes that lead to BL:

Answer 32

“Chromosomal Translocations:

1. That place the MYC gene under the regulation of the Ig heavy chain so there is c-myc dysregulation.
2. These are three distinct alternative chromosomal translocations involving chromosomes 2,14 and 22.
3. In all three translocations a region from one of these three chromosomes is fused to a section of chromosome 8.”

Question 33

How does Chromosome Translocation lead to Chronic Myeloid Leukaemia?

Answer 33

“Chromosomal Translocation generates the Philadelphia Chromosome:

1. This codes for the BCR-ABL Fusion Protein.
2. As a result of this translocation the tyrosine kinase activity of the oncogene ABL is constitutive.
3. Leading to abnormal proliferation.
4. 95% of CML patients carry the Philadelphia Chromosome.”

Question 34

What is the therapeutic strategy for CML?

Answer 34

“Imatinib (Gleevac) a Tyrosine Kinase Inhibitor.

96% remission in early stage patients.”

Question 35

How were Tumour Suppressor Genes discovered and identified?

Answer 35

“Somatic Cell Hybridization Experiments:

• Fusion of normal cells with tumour cells yielded hybrid cells containing chromosomes from both parents.
• These cells were not capable of forming tumours.
• Genes derived from the normal parent acted to inhibit or suppress tumour development.
• The first tumour suppressor gene was identified by studies of retinoblastoma a childhood eye tumour.”

Question 36

What are the 3 main functions of Tumour Suppressor Genes?

Answer 36

“1. Act as Stop Signs to uncontrolled growth.

2. Promote Differentiation.
3. Trigger Apoptosis.

Therefore they are usually regulators of cell cycle checkpoints.
e.g RB1, differentiation (APC) or DNA repair (BRCA1). “

Question 37

How are Tumour Suppressor Genes inactivated?

Answer 37

“Loss of tumour suppressor gene function requires inactivation of both alleles of the gene.
Inactivation can be a result of mutation or deletion.
They are defined as recessive genes.
Sometimes referred to as ‘anti-oncogenes’. “

Question 38

What is Retinoblastoma?

Answer 38

“Rare Childhood Cancer:

• Develops when immature retinoblasts continue to grow very fast and do not turn into Mature Retinal Cells.
• An eye that contains a tumour will reflect back light in a white colour (leukocoria).
• This is casued by a hereditary mutation on chromosome 13 the retinoblastoma gene.”

Question 39

What are the two forms of Retinoblastoma?

Answer 39

“Two forms of the disease:

• Familial 40%
• Sporadic 60%. “

Question 40

How can mutations lead to the development of Retinoblastoma?

Answer 40

“Development of Retinoblastoma requires two mutations.

Which are known to correspond to loss of both functional copies of the Rb gene.”

Question 41

What are Loss of Function Mutations?

Answer 41

“Loss-of-Function Mutations:

• Mutations that inactivate Tumour Suppressor Genes.
• These are often Point Mutations or Small Deletions.
• That disrupt the function of the protein that is encoded by the gene.”

Question 42

What is Loss of Heterozygosity?

Answer 42

“Loss of Heterozygosity:

• Leads to the inactivation of the second copy of a Tumour Suppressor Genes.
• This is known as the Two Hit Hypothesis.
• A Heterozygous cell receives a second hit in its retaining functional copy of the tumour suppressor gene.
• Thereby becoming Homozygous for the mutated gene. “

Question 43

Compare the mutations in a Hereditary against a Sporadic Tumour:

Answer 43

“Hereditary:

1. HIT: Inherited Mutation.
2. HIT: Acquired Mutation

Only 1 HIT on the Somatic Level.

Sporadic:

1. HIT: Acquired Mutation.
2. HIT: Acquired Mutation

2 HITS on the Somatic Level.”

Question 44

What are the 3 members of the RB gene family?

Answer 44

“1. Rb/p105/110,
2. p107
3. Rb2/p130
These are known as Pocket Proteins.”

Question 45

What is the Retinoblastoma Protein (pRB) and can it bind to?

Answer 45

“1. pRb is a multi functional protein with over 100 binding partners.

2. It is a transcriptional co factor that can bind to Transcription Factors.
3. The main binding partner is the E2F Transcription Factor which interacts with the large pocket.
4. Other Viral Oncoproteins can bind to Rb.”

Question 46

What are the Functions of Rb and how does it carry these out?

Answer 46

“Functions in diverse cellular pathways such as:

• Apoptosis.
• Cell Cycle.

It regulates these pathways through the stimulation or inhibition of the activity of interacting proteins.”

Question 47

What regulates the passage of a Cell through the Cell Cycle?

Answer 47

“1. Cyclins

2. Their associated CDKs”

Question 48

What is the main function of Rb and how does it do this?

Answer 48

“Main function of Rb is to regulate the cell cycle.

It does this by inhibiting the G1 to S Phase transition.”

Question 49

Describe the interaction between Cyclin D and Rb:

Answer 49

“1. Cyclin D is the first Cyclin to be synthesized

2. Cyclin D and CDKS4/6 both drive progression through G1.
3. A key substrate for Cyclin D is RB protein.
4. Cyclin D and E families and their CDKs phosphorylate Rb.
5. Rb activity is regulated by this phosphorylation, phosphorylated Rb is inactive.

G1 checkpoint leads to the arrest of the cell cycle in response to DNA damage.”

Question 50

How does Phosphorylation affect the activity of Rb?

Answer 50

“Rb protein regulates the activity of the E2F Transcription Factor which is needed for S Phase:

1. Dephosphorylated Rb:
   - Dephosphorylated Rb is Active and remains bound to E2F.
   - Active Rb blocks the progression of the Cell Cycle from the G1 to the S Phase.
   - So when the Rb Tumour Suppressor is Active it CAN Inhibit Cell Proliferation.
2. Phosphorylated Rb:
   - Phosphorylated/Hyperphosphorylated Rb is Inactive and releases E2F.
   - When E2F is released it migrates to the nucleus to induce transcription.
   - Inactive Rb cannot block the progression of the Cell Cycle from the G1 to the S Phase.
   - So when the Rb Tumour Suppressor is Inactive it CANNOT Inhibit Cell Proliferation. “

Question 51

In what ways can the Retinoblastoma Protein be inactivated?

Answer 51

“1. Phosphorylation.

2. Mutation.
3. Viral Oncoprotein Binding:
   - Viral inactivation is found in small DNA tumour viruses by disrupting E2F binding or destabilisation of Rb.
   - For Example:
   Adenovirus - E1A.
   Papilloma E7
   Polyoma - large T antigen
4. Retinoblastoma:
   - pRb is functionally inactivated by Mutations or Partial Deletions.”

Question 52

How is Rb Phosphorylation affected in Cancer?

Answer 52

“1. In cancer cells Rb Phosphorylation is deregulated throughout the Cell Cycle.

2. As a direct consequence E2F transcription factors can induce deregulation of the Cell Cycle.
3. Normally G1 checkpoint leads to the arrest of the Cell Cycle in response to DNA damage.
4. Without Rb on watch cells move through G1 into S and are not subjected to usual checks. “

Question 53

What is the P53 Tumour Suppressor?

Answer 53

”- First tumour suppressor gene to be identified.

• At the heart of the cell’s Tumour Suppressive Mechanism.
• It is involved in Sensing DNA Damage and regulating Cell Death/Apoptosis as well as other pathways.
• Specialises in preventing the appearance of abnormal cells.
• It can bind to around 300 different gene promoter regions and its main role is as a Transcription Factor. “

Question 54

Is there a link between P53 Mutation and Cancer?

Answer 54

“P53 is mutated in 30-50% of commonly occurring human Cancers.
Frequent mutation of p53 in Tumour Cell Genomes suggests that Tumour Cells try to eliminate p53 function before they can thrive.”

Question 55

List the main domains of the P53 Tumour Suppressor?

Answer 55

“1. Amino Transactivation Domain

2. Central DNA Binding Domain.
3. Tetramerization Domain.
4. Carboxyl Regulatory Domain “

Question 56

What is the MDM2 Protein?

Answer 56

“A Ubiquitin Ligase.

Also an Oncogene.”

Question 57

Describe how p53 is regulated by MDM2:

Answer 57

“1. Normally levels of p53 protein are low in cells.

2. These levels are kept low by MDM2 Protein.
3. In unstressed normal cells both p53 and MDM2 move between the Nucleus and Cytosol.
4. MDM2 binds to p53 to form a complex in the Nucleus.
5. In the Nucleus MDM2 modifies the Carboxyl Terminus of p53.
6. This targets the p53 for degradation by the Proteasome.
7. This is why p53 has a short 20 minute half life.”

Question 58

What external factors can activate p53?

Answer 58

“Stress Signals can activate p53:

1. Signals are sensed mainly by Kinases,
2. These Kinases then phosphorylate p53.
3. Phosphorylation of p53 disrupts the interaction between p53 and MDM2.”

Question 59

Give an example of stress signals activating p53:

Answer 59

“Ionizing Radiation Signals through two kinases: ATM/ATR.
Activate Oncogenes such as RAS.
Which induce activity of p14arf responsible for sequestering MDM2. “

Question 60

Which kinds of Genes can P53 regulate?

Answer 60

“Genes involved in:

• DNA Damage Repair.
• Apoptosis.
• Cell Cycle Arrest. “

Question 61

What is the significance of mutations in p53 dysfunction?

Answer 61

“Mutational Inactivation is one of the most popular mechanisms behind the dysfunction of p53.
More than half of human cancers carry loss of function mutations of p53.
Among them 95% of mutations were detectable within the DNA Binding Domain. “

Question 62

How can p53 be a good Therapeutic target for Cancers?

Answer 62

“As of p53 is a major protein in suppressing Tumorigenesis.
It is a promising Therapeutic Target for Cancer.
Strategies are aimed at correcting p53 mutation and restoring wild type p53 function.”

Question 63

How can Retroviruses be used in Gene Therapy for Cancer?

Answer 63

“Retroviruses integrate in a stable form into the genome of infected cells.
Retrovirus Mediated Gene Transfer:
- Of the wildtype TP53 gene into both Human Lung Tumour Cell Lines and Xenograft Models.
- Could lead to the inhibition of Tumour Cell Growth.”

Question 64

Give examples of how inhibitors could be used as Therapeutic strategy:

Answer 64

“1. PRIMA - 1 restores mutant p53 by modifying the thiol groups in the core domain of the protein.

2. Nutlin is a potent MDM2 antagonist
3. RITA binds to p53 and can restore mutp53 activity.
4. Inhibitors of CRM1 result in nuclear accumulation of p53.”

Question 65

How are all Tumours different?

Answer 65

“The genetics of every tumour is unique.

No two tumours will be identical.”

Question 66

What is Personalised Medicine?

Answer 66

“Tailoring the treatment according to the Genetics of the Tumor.
Avoids patients being subjected to unnecessary combinations of drugs which are unpleasant and won’t work.”

Question 67

Why is Genetic Analysis/Personalised Medicine much more effective?

Answer 67

“1. You get a detailed readout of the molecular faults in a patient’s tumour.

2. Classifies them according to the genetic make up instead of just their location in the body.
3. People with the ‘same’ cancer can have different forms of the disease so responses to treatment vary.
4. Cancers growing in different parts of the body may also share the same genetic faults so respond to similar treatments.”