Oncogenes and Tumour Suppresor Genes

Question 1

What are the 4 major functional changes in cancer ?

Answer 1

1. Increased growth - loss of growth regulation , stimulation of environment promotes 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 (chromosomal instability) = damaged cells not repaired+not removed by apoptosis

Question 2

What is an oncogene mutation and what can it lead to ?

Answer 2

Mutations in the genes that regulate cell growth + cell death (oncogenes + tumour suppressor genes).

Gain of function mutation -

Continual cell division = tumour

• An altered gene whose product can act in a dominant fashion to help make a cell cancerous
• Oncogene is a mutant form of a normal gene (proto-oncogene) involved in the control of cell growth or division

A single mutation in 1 oncogene allele = Activates oncogene = abnormal cell proliferation

Oncogenes = normal genes in cells that regulate cell growth in response to growth signals

Question 3

What is a tumour suppressor mutation and what can it lead to ?

Answer 3

Tumour suppressors cause cell cycle arrest, repair DNA damage/remove cell by apoptosis
This is loss of function -

A gene whose normal activity prevents formation of a cancer

Both alleles for the tumour suppressor must be mutated

1 mutation in 1 allele insufficient to completely knock out tumour suppressor function

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)

Loss of function mutation = cells proliferate abnormally

Question 4

What was Rous’s protocol for inducing sacroma in chickens ?

Answer 4

Sarcoma = bone/muscle tumour

1. Chicken with sarcoma in breast muscle
2. Rous Broke the sarcoma into small tissue chunks
3. Ground sarcoma with sand
4. Passed this through a filter to produce filtrate (removes bacteria)
5. Inject the filtrate into young healthy chicken
6. Observed sarcoma in the injected chicken

He concluded that a virus must be responsible for the induction of tumour formation.

Rous sarcoma virus

Question 5

How were retroviruses important experimentally ?

Answer 5

There is a Strong association of some viruses with cancer

Technological advances
Lots of Funding
Improved tissue culture techniques
The discovery of reverse transcriptase , RNA genome , replicates via DNA intermediate and they are enveloped

Question 6

What is V-Src (viral-Src)?

Answer 6

This was an (extra gene) oncogene found in the retrovirus genome.

V-src= proto oncogene altered from transduced by retroviruses.

It was concluded that the Rous sarcoma viral gene was a host gene (v-SRC = mutated cellular gene) which was ‘ kidnapped’ from the host cell and transformed into an oncogene.

An oncogene is any cellular gene that upon activation can transform cells.

c-SRC = cellular oncogene

Rous sarcoma virus infects chicken cell, goes through reverse transcription = end up w dsDNA provirus. RNA → DNA. provirus gets integrated next to host cellular SRC sequence, fusion, packaged into capsid = Rous sarcoma virus that contains SRC gene (kidnapped gene)

Question 7

Describe the process of capture of c-src by retrovirus

Answer 7

During evolution , the virus can acquire fragments of genes from the host at integration sites and this process results in the creation of oncogenes.

The oncogene product was characterised as a 60kDa intracellular tyrosine kinase.

Can phosphorylate cellular proteins and effect growth

Exception to central dogma DNA→RNA→protein. RNA→DNA

Question 8

Describe the typical retroviral life cycle

Answer 8

There is infection of the host cell. It will then undergo reverse transcription which produces dsDNA provirus .Following this there is accidental integration next to host c-src. The consequence is a fusion which gets packaging into capsid.
This will form a RSV virion which carries the src sequences

Question 9

What does the v-src oncogene cause ?

Answer 9

v-src oncogene is responsible for causing cancer

alteration to SRC sequence = important bc SRC controls cell growth. mutation = loss of ability to regulate this process. Rous sarcoma virus = kidnap this gene, ends up in chickens, causes abnormalities. but in human cells, these sequences are normal genes = proto-oncogenes. only when mutated = active oncogenes. oncogene is activated by exposure to carcinogens (model 1 - chemical/physical/viral). active oncogene which has gained function = problem

Through hybridisation experiments , they found that the c-src gene was present in the genomes 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 , 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.They become active oncogenes following mutation

Various agents including radiation, chemical carcinogens and exogenously added viruses may transform cells by switching on the endogenous oncogenic information.

Question 10

Describe Viral oncogenesis and how it can be transmitted

Answer 10

15-20% of human cancers are caused by oncoviruses.

Viral oncogenes can be transmitted by either DNA /RNA viruses.

DNA viruses can cause lytic infection which can lead to death of the cellular host or can replicate their DNA along with that of the host and promote neoplastic transformation.

Viruses themselves also have oncogenes = express viral oncogenic proteins. when end up in host cell, cause transformation of host cell.

Question 11

Describe DNA viruses

Answer 11

DNA viruses- These encode various proteins along with environmental factors can initiate and maintain tumours.

Question 12

Describe RNA viruses

Answer 12

RNA viruses - Integrate DNA copies of their genomes into the genome of the host cell and as these contain transforming oncogenes they can induce cancerous transformation of the host.

Question 13

Describe the process of oncogene activation

Answer 13

Oncogenes for every type of protein involved in growth factor signal transduction pathway

Activation of oncogene is usually by altering the genomic sequence of the gene. (Mutations, insertions, amplifications and translocations)

=Loss of ability to respond to growth factors

Proto-oncogene becomes an oncogene

Question 14

What are different ways in which oncogenes can be activated ?

Answer 14

There is a protein encoded by proto oncogene:

a. Point mutation/deletion/amplification/duplication/translocation which causes an encoded protein with altered structure/function
b. Gene duplication which can cause increased synthesis of encoded protein = activates gene
c. DNA regulatory sequence translocated from distant site alters expression of downstream gene which causes increased synthesis of encoded proteins , synthesis of protein containing portions encoded by different genes, protein-coding gene translocated from distant site fuses with portion of gene causing formation of a fusion gene

Various oncogenes are activated in diff. ways to form diff. tumour types. e.g. L-myc is activated by amplification in lung carcinoma

Question 15

What are the 4 types of proteins which are involved in the transduction of growth signals?

Answer 15

Proto-oncogenes encode components of the growth factor signal transduction pathways and can be 1 of the 4 following proteins:

• Growth factors e.g.EGF
• Growth factor receptors ErbB
• Intracellular signal transducers
• Intracellular signalling molecules e.g. Ras/Raf
• Nuclear transcription factors - translocate to nucleus

EGF binds to ErbB Receptor. Then recruits lots of Ras/Raf molecules, signalling cascade, molecules enter nucleus = specific gene transcription

Disrupt signalling pathway = oncogene. Cell proliferation pathways

Question 16

How do oncogenes act as growth factors ?

Answer 16

Most oncogenes proteins function as elements of the signalling pathways that regulate cell proliferation and survival in response to growth factor stimulation.

Oncogene proteins act as growth factors e.g. EGF, growth

Question 17

What are the functions of Ras/Raf ?

Answer 17

Ras/Raf activates the ERK MAP kinase pathway, inducing additional genes (fos) that encode potentially oncogenic transcriptional regulatory proteins.

Question 18

Give an example of an intracellular signal transducer

Answer 18

RAS proteins are small GTPases that are normally bound to GDP in inactive state. RAS oncogene = GTPase.

Oncogenic activation of ras is seen in around 30% of human cancers.

Activated through mutations.
Codons 12,13,61
-Glycine → valine - Bladder carcinoma (codon 12)
-Glycine → cysteine - Lung cancer

Mutation activates the oncogene

Question 19

What is the normal function of RAS (Intracellular Signal Transducer?

Outline its mechanism .

Answer 19

Intracellular signal transducers

1. Binding of extracellular growth factor signal to Receptor
2. Promotes recruitment of RAs (GTPase) 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 e.g. transcription factor to promote expression of proteins important for growth and survival (regulate cell cycle).

Ras hydrolyses GTP to GDP quickly which switches itself off

Question 20

Describe the difference in this mechanism in ‘hyperactive ‘ Ras (mutation)

Answer 20

There is a point mutation in codons 12,13 and 61

The consequence of each of these mutations is a loss of GTPase activity of the RAS protein normally required to return active RAS to the inactive RAS GDP.

This leads to constitutive activation

Hyperactive Ras protein (product of oncogene) issues signals on its own.Active oncogene, Gain of function, always switched on, cells continuously dividing + progressing through cell cycle = tumorigenesis

Question 21

Outline the MYC oncogene family + its function

Answer 21

MYC = Transcription Factor, regulates various processes

The MYC oncogene family consists of 3 members :
C-MYC, MYCN and MYCL which encodes c-Myc, N-MYC and L-MYC

• Originally identified in avian myelocytomatosis virus (AMV)
• 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

MYC is activated by chromosomal translocation (not by mutation)

Question 22

MYC is activated by …………..

Answer 22

MYC is activated by chromosomal translocation (not by mutation)

Question 23

Outline all the functions of the MYC family of genes

Answer 23

Signal transduction
Cell cycle
Metabolism
Translation
DNA repair
Cell adhesion /cytoskeleton
MicroRNAs
Protein biosynthesis
Transcription

Question 24

Outline the mechanism through which MYC is activated

Answer 24

The Myc oncogene is overexpressed in majority of human cancers and can causes at least 40% of cancers

Encodes a helix-loop-helix leucine zipper transcription factor that dimerises with its partner protein - Max= transactivates gene expression

Activated when it comes under control of foreign transcriptional promoters.
Leads to deregulation of the oncogene which drives proliferation.

This is a result of chromosomal translocation

Question 25

Outline the mechanism of activation of MYC in Burkitt’s Lymphoma (BL)

Answer 25

Epstein Barr virus causes Burkitt’s Lymphoma (tumour)

BL cases carry 1 of 3 characteristic chromosomal translocations that place the MYC gene under the regulation of the Ig heavy chain.
c-myc expression is deregulated

In BL, 3 chromosomal translocations occur involving 2,14,22

A region from 3 chromosomes is fused to a section of chromosome 8 = lose control of Myc = proliferation

Question 26

Give an additional example of chromosomal translocation that activates an oncogene

Answer 26

Chronic myelogenous leukaemia which accounts for 15-20% of all leukaemias.

95% of CML patients carrying the **philadeliphia chromosome** which is the product of the chromosomal translocation t(9;22)(q34;q11). 
ABL oncogene (chr9) + BCR oncogene (chr22) fuse

This causes a BCR-ABL fusion protein

ABL = tyrosine kinase

Result:
Tyrosine kinase activity of the oncogene ABL is constitutive, leading to abnormal proliferation.

Therapeutic strategies for CML include imatinib (Gleevac) = a tyrosine kinase inhibitor

Question 27

What are tumour suppresor genes?

Answer 27

Tumour suppresor genes control the processes which regulate cell numbers

Tumour suppressor gene products act as stop signals to uncontrolled proliferation .
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 (= recessive genes/anti-oncogenes)

Question 28

What is Retinoblastoma ?

Answer 28

A rare childhood cancer which develops when immature retinoblasts continue to grow fast and do not turn into mature retinal cells.

Retinoblasts fail to mature = tumour in back of eye.

Leukocoria

2 Types:
Familial (40%) / Sporadic (60%)

Mutation is on chromosome 13 (13q14) , the retinoblastoma 1 (Rb1) gene

Question 29

How was Retinoblastoma discovered?

Answer 29

Proposed :
Development of retinoblastoma requires 2 mutations (1 in each allele) which cause loss of the functional copies of the Rb gene = 2 hit hypothesis (Knudson)

For cell to wipe out function of the tumour suppressor, it has to target both alleles independently

slide 34

Question 30

What is loss of heterozygosity?

Define this

Answer 30

Loss of heterozygosity describes the process that leads to the inactivation of the second copy of a tumour suppressor gene , a heterozygous cell receives a second hit in its remaining functional copy of the tumour suppressor gene = becomes homozygous for the mutated gene

Mutations that cause this are called loss-of-function mutations.
Point mutations/small deletions which disrupt the function of the protein that is encoded by the gene

Question 31

Outline the Retinoblastoma protein structure

Answer 31

The Rb gene group includes : Rb/(p105/110), p107 and Rb2/p130
(These are called pocket proteins).

pRb is a multifunctional protein (110kDa).
(A transcriptional co-factor which can bind to transcription factors )

RB’s large pocket area interacts with E2F transcription factor

Rb = transcriptional cofactor

Question 32

How is Rb involved in the cell cycle ?

Answer 32

Rb regulates the cell cycle by inhibiting the G1 → S phase transition .

2 important proteins involved in the cell cycle are :
cyclins and their associated cyclin dependent kinase (cdks)

Passage of a cell through the cell cycle is regulated cyclins and cyclin dependant kinases (cdks)

slide 36

Question 33

What is the first cyclin which is synthesised in the cell cycle ?

Answer 33

Cyclin D is the first cyclin to be synthesised and drive progression through G1 together with cdks4/6.

The G1 checkpoint leads to the arrest of the cell cycle to allow to repair DNA damage .

Substrate for cyclin D is Rb protein

Cyclin D + E families and their CDKs phosphorylate RB

Question 34

What are the consequences of phosphorylated Rb?

Answer 34

Rb regulates G1 → S transition

Rb protein regulates the activity of the E2F transcription factor crucial for the expression of genes required for the S phase.

Rb activity is regulated by phosphorylation

When the Rb tumour suppressor is active, it inhibits cell proliferation. hypophosphorylated form.

When Rb is dephosphorylated /hypo phosphorylated it is active and binds to E2F

When Rb is active, it blocks the progression G1 → S phase. encourages cell cycle arrest.

When Rb is hyperphosphoryted , in response to extracellular physiological signals it is inactive

extracellular signals, damage = phosphorylation of Rb due to cyclins = E2F no longer binds to phosphorylated Rb.

active Rb tumour suppressor = Rb regulates, keeps E2F bound to it. if Rb is phosphorylated = becomes inactivated = releases E2F = E2F translocates into nucleus = cell cycle progression, no cell cycle arrest

Inactivate by phosphorylation

Question 35

What other methods inactivate Rb leading to loss of function ?

Answer 35

Mutation, Viral Oncoprotein Binding.

Viral infections give rise to oncogenes that destabilise E2F/Rb 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 destabilisatiation of Rb
Adenovirus-E1A
Papilloma-E7
Polyoma-Large T antigen

In cancer cells RB phosphorylation is deregulated throughout cell cycle .E2F transcription factors can induce deregulation of the cell cycle .
Cells will therefore move through G1- S without further checks

Virus causes cell to transform by tampering w tumour suppressor function = continuous cell proliferation. No Rb = continual G1→S

Question 36

What is p53 and what is its function ?

Answer 36

The p53 gene was the first tumour suppressor gene to be identified.

p53 protein = ‘guardian of the genome’

Senses DNA damage and regulates cell death/apoptosis

p53 is mutated in 30-50% of cancers

Prevents appearance of abnormal cells

p53 = TF, responds to diff. signals

Question 37

What is the structure of p53?

Answer 37

It is a transcription factor

Has a amino transactivation domain , central DNA-binding domain , tetramerization domain and a carboxy regulatory domain.

Can bind to around 300 gene promoter regions - Transcription factor

Question 38

What is the normal regulation of p53 ?

Answer 38

Normally low levels of p53 proteins

Kept low by MDM2 protein = a ubiquitin ligase. adds ubiquitin onto lysine residues, then is targeted to proteasome for degradation

In unstressed normal cells both p53 and MDM2 move between nucleus and cytosol.

MDM2 binds p53 to form a complex in the nucleus where MDM modifies the carboxyl terminus of p53 and targets it for degradation by the proteasome .

WT p53has a short 20 min half life

Question 39

How is p53 activated ?

Answer 39

Activation of p53 tumour suppressor

Stress signals are able to activate p53

Signals are sensed by kinases which will phosphorylate the p53.

This will disrupt the interaction between it and MDM2.

Ionising radiation signals through two kinases ATM/ATR activate oncogenes such as ras induce activity of p15arf responsible for sequestering MDM2.

p53 can therefore regulate genes involved in DNA Damage repair apoptosis and cell cycle arrest

phosphorylate p53 via CHEK1/CHEK2 , then phosphorylate p53 = translocates into nucleus + regulates processes involved in cell cycle arrest, DNA repair, apoptosis

wildtype p53 regulates DNA damage mechanisms

Question 40

many p53 mutations tamper with its ………….

Answer 40

DNA binding ability

= p53 is good target for therapy

Question 41

Outline the therapeutic strategies which have been used to treat p53 mutations

Answer 41

Retroviruses integrate in a stable form into the gnome of infected cells .

Retrovirus-mediated gene transfer of the wild-type TP53 gene into human lung tumour cell lines and xenograft models could lead to inhibition of tumour cell growth.

1. PRIMA-1 restores mutant p53 by modifying the thiol groups in the core domain of the protein = refolds back into wildtype p53
   2.Nutlin = potent MDM2 antagonist (enhances half life) to keep in cell for longer, prevents ubiquitination+degradation of p53????
   3.RITA binds to p53 and can restore mutp53 activity
   4.Inhibitors of CRM1 result in nuclear accumulation of p53
   (inhibits export of p53 into cytoplasm to proteasome for degradation, keeps it in nucleus to function as tumour suppressor)

• put wildtype p53 back into tumour cells
• use inhibitors: -refold mutant p53 back into wildtype. -regulate p53 regulators (e.g. MDM2) = greater p53 halflife = greater tumour suppressor function.

Question 42

Which genes drive proliferation?

Answer 42

Oncogenes = normal genes in cells that regulate cell growth in response to growth signals

Question 43

Which top 2 hallmarks are relevant to oncogenes + tumour suppressor genes?

Answer 43

• Lost ability to control proliferation - not responding to growth signals in the same way
• Evade growth suppressors

Question 44

Oncogenes -
Tumour suppressors -

Answer 44

Oncogenes - Proliferation
Tumour suppressors - Apoptosis

Question 45

2 major types of mutated gene contribute to carcinogenesis
Oncogene mutation ……….. the growth process
Tumour suppressor mutation ………………. of car

Answer 45

Oncogene normal function = drives cell division. Oncogene mutation speeds up = accelerator of car. Gain of function mutation.

Tumour suppressor normal function = counteracts oncogene mutation. Tumour suppressor mutation inactivate the brakes of car. Loss of function mutation

Question 46

SRC =

Answer 46

SRC = cellular gene

Question 47

Discovery of Tumour Suppressor Genes

Answer 47

Somatic cell hybridisation. Fuse normal healthy cell + tumour cell = nontumorigenic hybrid cell = a component of normal cell suppresses tumour growth

First tumour suppressor gene discovered = Rb

Tumour suppressor genes counterbalance oncogenes.

Oncogenes target cell proliferation + survival

Tumour suppressor genes repair mutation, remove damaged cells (apoptosis).

Tumour suppressors regulate these various processes

Question 48

Loss of function of a tumour suppressor is associated with ……..

Answer 48

various tumours

p53 is mutated in half of all tumours