Oncogenes 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 cell death (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 are the two major types of mutated genes that contribute to carcinogenesis?

Answer 2

Oncogenes:
- Their normal job is to make cells divide, driving cell division forward
- In cancer, pick up mutations that mean they are permanently active – a bit like putting a brick on the accelerator. The car approaches the red light and can’t stop
- Oncogene: “Gain of function”
Tumour Suppressor Genes
- Tumour suppressor genes are like the car’s brakes.
- Even if you have a mutation in an oncogene that pushes cell division forward, if your tumour suppressor genes are strong enough, they should still be able to counteract the oncogene
- In cancer, pick up mutations that switch the gene off. This is like cutting the brakes in a car. Even if there is no oncogenic brick on the accelerator, without breaks the car definitely can’t stop
- Tumour Suppressor gene: “Loss of function”

Question 3

What is Rous’s protocol for inducing sarcoma in chickens?

Answer 3

1. Chicken with sarcoma in breast muscle
   
   2. Remove sarcoma and break it up into small chunks of tissue
   3. Grind up sarcoma with sand
   4. Collect filtrate that has passed through fine-pore filter
   5. Inject filtrate into young chicken
   6. Observe sarcoma in injected chicken
      Tumours developed weeks later
      Taking the new sarcoma, filtrates produced could also induce tumours in other chickens
      The cycles could be repeated indefinitely. Also 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
      Rous concluded that a virus must be responsible for the induction of tumour formation
      Discovery that this sarcoma was transmissible through viruses- Rous Sarcoma Virus

Question 4

How was the c-src gene captured?

Answer 4

During evolution, the virus can acquire fragments of genes from the host at integration sites and this process results in the creation of oncogenes
Somehow the provirus is somehow accidentally integrated next to the host cellular src sequence, you end up with a fusion and that then gets packaged into the capsid
And you end up with a rous sarcoma virus that contains the src gene

Question 5

What is the oncogene hypothesis?

Answer 5

Bishop and Varmus used different strains of Rous sarcoma virus in their research, they:
Identified the v-src oncogene as responsible for causing cancer.
Used hybridization experiments, and they found that the c-src gene was present in the genome of many species.
They then showed that the host cell c-src gene was normally involved in the positive regulation of cell growth and cell division.
Following infection, however, the v-src oncogene was expressed at high levels in the host cell, leading to uncontrolled host cell growth, unrestricted host cell division, and cancer.
Proto oncogenes are normal genes that can control growth
Various agents, including radiation, chemical carcinogens, and, perhaps, exogenously added viruses, may transform cells by “switching on” the endogenous oncogenic information.

Question 6

What is viral oncogenesis?

Answer 6

Viral oncogenes can be transmitted by either DNA or RNA viruses.
DNA Viruses:
- Encode various proteins along with environmental factors can initiate and maintain tumours
RNA Viruses
- Integrate DNA copies of their genomes into the genome of the host cell and as these contain transforming oncogenes that induce cancerous transformation of the host

Question 7

How are some oncogenes activated?

Answer 7

There are examples of oncogenes for every type of protein involved in a growth factor signal transduction pathway
These genes captured by animal retroviruses are altered in human cancer, activation can involve
- mutations, insertions, amplifications, and translocations
This leads to loss of response to growth regulatory factors,
Only one allele needs to be altered

Question 8

How do proto-oncogenes encode components of the growth factors signal transduction pathway?

Answer 8

Proto-oncogenes can be 1 of 4 types of proteins are involved in the transduction of growth signals
Normally:
- Growth factors
- Growth factor receptors
- Intracellular signal transducers
- Nuclear transcription factors
Growth factors, signal transduction and cancer
- The majority of oncogene proteins function as elements of the signalling pathways that regulate cell proliferation and survival in response to growth factor stimulation

Question 9

What is the RAs oncogene family?

Answer 9

RAS genes were identified from studies of two cancer-causing viruses the Harvey sarcoma virus and Kirsten sarcoma virus
RAS proteins are small GTPases that are normally 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
E.g. codon 12; glycine to valine gives rise to bladder carcinoma

Question 10

How is Ras activated and what happens when it is activated?

Answer 10

Intracellular Signal Transducers
1. Binding of extracellular growth factor signal
2. Promotes recruitment of RAS proteins to the receptor complex
3. Recruitment promotes Ras to exchange GDP (inactive Ras) with GTP (active Ras)
4. Activated Ras then initiates the remainder of the signalling cascade (mitogen activated protein kinases)
5. These kinases ultimately phosphorylate targets, such as transcription factor to promote expression of genes important for growth and survival
Normally Ras hydrolyzes GTP to GDP fairly quickly, turning itself “off”
A point mutation will cause a hyperactive Ras

Question 11

What is the MYC oncogene family?

Answer 11

The MYC oncogene family consists of 3 members, C-MYC, MYCN, and MYCL, which encode c-Myc, N-Myc, and L-Myc, respectively
The MYC oncoproteins belong to a family of transcription factors that regulate the transcription of at least 15% of the entire genome
Major downstream effectors of MYC include those involved in ribosome biogenesis, protein translation, cell-cycle progression and metabolism, orchestrating a broad range of biological functions, such as cell proliferation, differentiation, survival, and immune surveillance (Basically its involved in a lot of things)

Question 12

What does the MYC oncogene family do and how is it linked to cancer?

Answer 12

The MYC oncogene is overexpressed in the majority of human cancers and contributes to the cause of at least 40% of tumours
It encodes a helix-loop-helix leucine zipper transcription factor that dimerizes with its partner protein, Max, to transactivate gene expression
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 13

How is MYC activated in Burkitt’s lymphoma?

Answer 13

Activation of MYC in Burkitt’s Lymphoma
Epstein Barr virus is associated with Burkitt’s lymphoma (BL)
BL is a high grade lymphoma that can effect children from the age of 2 to 16 years
In central Africa, children with chronic malaria infections have a reduced resistance to the virus.
All BL cases carry one of three characteristic chromosomal translocations that place the MYC gene under the regulation of the Ig heavy chain.
Therefore c-myc expression is deregulated
In BL, three distinct, alternative chromosomal translocations involving chromosomes 2, 14 and 22
In all three translocations a region form one of these three chromosomes is fused to a section of chromosome 8

Question 14

How is chromosomal translocation responsible for Chronic Myelogenous Leukaemia (CML)?

Answer 14

95% of CML patients carry the Philadelphia chromosome, that is the product of the chromosomal translocation generating the BCR-ABL fusion protein
As a result of this translocation the tyrosine kinase activity of the oncogene ABL is constitutive leading to abnormal proliferation
Therapeutic strategies for CML include Imatinib (Gleevac) a tyrosine kinase inhibitor-96% remission in early-stage patients

Question 15

What do tumour suppressor genes do?

Answer 15

Body has mechanisms to ‘police’ processes that regulate cell numbers
Tumour suppressor gene products act as stop signs to uncontrolled growth, promote differentiation or trigger apoptosis
Therefore they are usually regulators of cell cycle checkpoints (e.g. RB1), differentiation (e.g. APC) or DNA repair (e.g. BRCA1)
Loss of tumour suppressor gene function requires inactivation of both alleles of the gene
Inactivation can be a result of mutation or deletion
Tumour suppressor genes are defined as recessive genes
Sometimes referred to as ‘anti-oncogenes’

Question 16

What is the retinoblastoma gene and how is it linked to retinoblastoma?

Answer 16

Retinoblastoma is a rare childhood cancer (1 in 20,000) that 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 light back in a white colour.
Often called a “cat’s eye appearance,” the technical term for this is leukocoria.
Two forms of the disease, familial (40%) and sporadic (60%)
The hereditary mutation is on chromosome 13 (13q14), the retinoblastoma 1 (Rb1) gene

Question 17

What is the structure of the retinoblastoma protein like ?

Answer 17

The Rb gene family includes three members: Rb/(p105/110), p107 and Rb2/p130 -collectively known as pocket proteins
pRb is a multifunctional protein with over 100 binding partners
A transcriptional cofactor that can bind to transcription factors
RB functions in diverse cellular pathways, such as apoptosis and the cell cycle, it has also become clear that RB regulates these pathways through the stimulation or inhibition of the activity of interacting proteins.
It’s main binding partner is the E2F transcription factor, interacting with the large pocket
Other viral oncoproteins can bind to Rb

Question 18

What is the main function of Rb?

Answer 18

Main function of Rb is to regulate the cell cycle by inhibiting the G1 to S phase transition
2 important proteins involved in the cell cyle are:
- Cyclins and their associated cyclin dependent kinases (cdks)
Passage of a cell through the cell cycle is regulated cyclins and cyclin dependent kinases (cdks)

Question 19

How are cyclins involved with Rb?

Answer 19

Cyclin D is the first cyclin to be synthesized and drive progression through G1 together with cdks4/6
The G1 checkpoint leads to the arrest of the cell cycle in response to DNA damage
A key substrate for cyclin D is RB protein
Cyclin D and E families and their cdks phosphorylate RB

Question 20

What happens to the retinoblastoma protein in the cell cycle?

Answer 20

Rb protein regulates the activity of the E2F transcription factor crucial for the expression of genes required for S phase
Rb activity is regulated by phosphorylation, it normally hypophosphorylated
When the Rb tumour suppressor is active it can inhibit cell proliferation
When Rb is dephosphorylated/hypophosphorylated it is active and remains bound to E2F
When Rb is active it blocks the progression of to S phase
When Rb is hyperphosphorylates , in response to extracellular physiological signals it is inactive
Upon phosphorylation of RB, E2F is released and migrates to the nucleus to induce transcription
When RB is inactive cell cycle progression from G1 to S occurs

Question 21

How does loss of function of Rb happen and what are the consequences?

Answer 21

Rb can be inactivated by phosphorylation, mutation, or viral oncoprotein binding
In retinoblastoma, pRb is functionally inactivated by mutations or partial deletions
Viral inactivation found in small DNA tumour viruses mainly by disrupting E2F binding or destabilisation of Rb
- Adenovirus produce E1A
- Papilloma produce E7
- Polyoma produce Large T antigen
All of which mess with Rb
In cancer cells RB phosphorylation is deregulated throughout cell cycle.
As a direct consequence E2F transcription factors can induce the deregulation of the cell cycle
Without RB on watch , cells move through G1 into S and are not subjected to usual checks

Question 22

What is the P53 Tumour Suppressor?

Answer 22

The p53 protein is at the heart of the cell’s tumour suppressive mechanism and has been nicknamed the ‘guardian of the genome’
It is involved in sensing DNA damage and regulating cell death/apoptosis as well as other pathways
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
p53 specializes in preventing the appearance of abnormal cells

Question 23

How is P53 regulated?

Answer 23

Normally levels of p53 protein are low in cells
These levels are kept low by MDM2 protein, a ubiquitin ligase (also an oncogene)
In unstressed normal cells both p53 and MDM2 move between the nucleus and cytosol
MDM2 binds p535 to form a complex in the nucleus where MDM modifies the carboxyl terminus of p53 and targets it for degradation by the proteasome
WT p53 has a short 20 min half life

Question 24

How is P53 activated?

Answer 24

Stress signals are able to activate p53
Signals are sensed by mainly kinases that then phosphorylate p53
Phosphorylation of p53 disrupts the interaction between it and MDM2 e.g. ionizing radiation signals through two kinases ATM/ATR activate oncogenes such as ras induce activity of p14arf responsible for sequestering MDM2.
P53 can thus regulate genes involved in DNA damage repair, apoptosis and cell cycle arrest

Question 25

What happens when P53 is mutated?

Answer 25

Mutational inactivation is considered to be one of the most common molecular mechanisms behind the dysfunction of p53.
Extensive mutation search revealed that 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
Role of p53 as a star player in suppressing tumorigenesis makes it a promising therapeutic target
Different strategies aimed at:
- Correcting p53 mutation and restoring wild-type p53 function by targeting its regulators

Question 26

What sort of therapeutic strategies can use these genes?

Answer 26

Gene therapy obvious approach
Many vectors and retroviruses have been examined
Retroviruses integrate in a stable form into the genome of infected cells. It has been demonstrated that retrovirus-mediated gene transfer of the wild-type TP53 gene into both human lung tumour cell lines and xenograft models could lead to the inhibition of tumour cell growth
Alternative strategies- use of inhibitors examples
- PRIMA-1, Restores mutant p53 by modifying the thiol groups in the core domain of the protein
- Nutlin- is a potent MDM2 antagonist preventing ubiquitination of p53
- RITA binds to p53 and can restore mutp53 activity
Inhibitors of CRM1 result in nuclear accumulation of p53

Question 27

How can we use genetic analysis for personalised medicine?

Answer 27

A detailed readout of the molecular faults in a patient’s tumour, and new generation of drugs that precisely target them
Classifies tumours according to their genetic make-up instead of where they grow in the body
People with the ‘same’ cancer can have different forms of the disease so responses to treatment vary
Cancers growing in different parts of the body may also share the same genetic faults so respond to similar treatments