Oncogenes and tumour suppressor genes

Question 1

What have tumour cells lost their ability to do?

Answer 1

→ The ability to control proliferation so they don’t respond to growth signals in the same way
→ You cannot suppress the growth of tumour cells.

Question 2

What major functional changes occur in cancer?

Answer 2

→ Increased growth (loss of growth regulation, stimulation of environment promoting growth e.g. angiogenesis)
→ Failure to undergo programmed cell death (apoptosis) or senescence
→ Loss of differentiation (including alterations in cell migration and adhesion)
→ Failure to repair DNA damage (including chromosomal instability)

Question 3

What do oncogenes do normally and what changes in cancer?

Answer 3

Oncogenes are like a car’s accelerator pedal.
→ 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: The mutation is a “Gain of function”
→ 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
(a “proto-oncogene”) involved in the control of cell growth or division.
→ A single mutation in one of the alleles of an oncogene is usually enough to activate that oncogene so you end up with cells proliferating abnormally

Question 4

What is the role of tumour suppressor genes and how does this change in cancer?

Answer 4

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, they 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: the mutation is a “Loss of function”
→ With tumour suppressors, you have to have a mutation in BOTH alleles - not just a mutation in one allele like oncogenes.
→ Then cells proliferate abnormally as a result.

Tumour Suppressor gene:
→ A gene whose normal activity prevents formation of a
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 5

What is a sarcoma?
How was this investigated initially?

Answer 5

A malignant tumour of connective or other non-epithelial tissue

→ A rare type of cancer that grow in connective tissue like bones, nerves, muscles, tendons, cartilage and blood vessels of the arms and legs

There was a scientist - Rous and he was bought a chicken with sarcoma tumour and the scientist cut the tumour and split it into small pieces and filtered it etc and injected into young chickens who also developed sarcoma. But he got rid of all bacteria etc so discovered that this sarcoma was transmissible through viruses-
Decades later oncogenic transformation by this virus was found to be caused by an extra gene contained in its genome an ‘oncogene’ called v-src

By 1976, homologous sequences were found in uninfected chickens and other organisms-
fruit flies to humans

Fundamental principle:
→ Oncogenes are alerted forms of normal genes or proto-oncogenes
→ c-src, cellular oncogene
→ v-src proto oncogene altered form transduced by retroviruses
→ Upon finding there was a gene homologous sequence to v-src in uninfected chickens, in 1989 Harold Varmus and J. Michael Bishop received the noble prize for laying down the foundation of mutations in carcinogenesis
→ Discovered that the some genes of cancer causing viruses were mutated forms of the cellular gene not viral genes
→ They concluded that the Rous sarcoma viral gene was in fact a host gene that had been ‘kidnapped’ by the virus (and ‘transformed’ into an oncogene)
→ An oncogene is any cellular gene that upon activation can transform cells
→ 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
→ So this is an exception- you go from RNA to DNA rather than the usual other way around. You end up with a RSV virion carrying src sequences.

Question 6

What did Bishop and Varmus continue to discover about oncogenes etc?

Answer 6

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.
→ It’s only when the proto oncogenes become mutated, we then refer to them as oncogenes/

Question 7

What 2 types of viruses can viral oncogenes be transmitted by?

Answer 7

→ Approximately 15%-20% of all human cancers are caused by oncoviruses
→ Viral oncogenes can be transmitted by either 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

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 they induce cancerous transformation of the host

Question 8

How many oncogenes have been identified to date?

Answer 8

→ To date-over 100 identified oncogenes
→ 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 a Loss of response to growth regulatory factors just ONE allele needs to be altered

Question 9

What are 4 types of proteins are involved in the transduction of growth signals? Give an example

Answer 9

1. Growth factors
2. Growth factor receptors
3. Intracellular signal transducers
4. 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
→ Oncogene proteins act as growth factors (e.g.EGF),
growth factor receptors (e.g. ErbB) and 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 10

What do we know about the Ras Oncogene family?

Answer 10

RAS Oncogene Family

→ RAS genes were identified from studies of two cancer-causing viruses the Harvey sarcoma virus and Kirsten sarcoma virus
→ These viruses were discovered originally in rats hence the name Rat sarcoma
→ 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. Glycine to valine - bladder carcinoma
Glycine to cysteine - lung cancer

Question 11

What is the normal function of the RAS Oncogene Family?

Answer 11

→ Binding of extracellular growth factor signal
→ Promotes recruitment of RAS proteins to the receptor complex
→ Recruitment promotes Ras to exchange GDP (inactive
Ras) with GTP (active Ras)
→ Activated Ras then initiates the remainder of the
signalling cascade (mitogen activated protein kinases)
→ These kinases ultimately phosphorylate targets, such as transcription factor to promote expression of genes
important for growth and survival Ras hydrolyzes GTP to GDP fairly quickly, turning itself “off”

Question 12

what happens when there is a Point mutation in codons 12, 13 and 61? How does it affect Ras?

Answer 12

→ You get hyperactive Ras
→ 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
→ There’s no stop to the cell cycle- cells are continuously dividing!

Question 13

What do we know about the transcription factor MYC? and the MYC oncogene?

Answer 13

→ The MYC oncogene family consists of 3 members,
C-MYC, MYCN, and MYCL, which encode c-Myc, N-Myc,
and L-Myc, respectively
→ 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
→ 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 of MYC is a result of chromosomal translocation- NOT a mutation

Question 14

What do we know about the activation of MYC in Burkitt’s Lymphoma?

Answer 14

→ 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. This is known as classical African or endemic BL
→ 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 15

What do we know about the Philadelphia chromosome?

Answer 15

→ Chronic myelogenous leukaemia (CML) accounts for 15-20% of all leukaemias
→ 95% of CML patients carry the Philadelphia chromosome, that is the product of the chromosomal translocation t(9;22)(q34;q11) 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 16

What do we know overall about tumour suppressor genes?

Answer 16

→ 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 17

What is retinoblastoma? What do we know about the retinoblastoma gene?

Answer 17

→ 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

→ The existence of the RB1 gene was predicted in 1971 by Alfred Knudson
→ Whilst studying the development of retinoblastoma he proposed that the development of retinoblastoma requires two mutations, which are now known to correspond to the loss of both of the functional copies of the Rb gene - “two-hit” hypothesis “Loss of heterozygosity“ often used to describe
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, thereby becoming homozygous
for the mutated gene.
→ Mutations that inactivate tumour suppressor
genes, called loss-of-function mutations, are
often point mutations or small deletions that
disrupt the function of the protein that is
encoded by the gene

Question 18

What do we know about the Retinoblastoma Protein RB Structure?

Answer 18

→ The Rb gene family includes three members: Rb/(p105/110), p107 and Rb2/p130 - collectively known as pocket proteins
→ pRb is a multi functional protein (110kDa) with over 100 binding partners
→ A transcriptional co factor 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.
→ Therefore, an important starting point for understanding RB function is its structure, which acts as a scaffold for these multiple protein interactions
→ It’s main binding partner is the E2F transcription factor, interacting with the large pocket
→ Other viral oncoproteins can bind to Rb
→ 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)
→ 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 19

For which transcription factor does the Rb protein regulate the activity of?

Answer 19

→ 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
→ 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 20

How can Rb be inactivated?

Answer 20

→ 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
1. Adenovirus - E1A
2. Papilloma - E7
3. Polyoma – Large T antigen

→ 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 21

What role does p53 have as a tumour suppressor?

Answer 21

→ The p53 gene was the first tumour suppressor gene to be identified
→ 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
→ The p53 protein has an amino transactivation domain, a central DNA binding domain, a tetramerization domain and a carboxyl regulatory domain
→ Can bind to around 300 different gene promoter regions-main role as a transcription factor

Question 22

What happens during regulation of P53 by MDM2?

Answer 22

→ 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 23

How can p53 be activated?

Answer 23

→ 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 24

p53 mutation/therapeutic strategies

Answer 24

→ 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 a s 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