16. Oncogenesis and Tumour Supressor Genes Flashcards
What are some major functional changes that occur in cancer?
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)
How do oncogenes and tumour suppressor genes (the two major types of mutated genes) work?
- Many oncogenes are normally components of growth factor signalling pathways that when mutated produce products in higher quantities or whose altered products have increased activity and therefore act in a dominant manner.
- In cancer, they pick up mutations that mean they are permanently active.
•ONCOGENES = “Gain of function” - Many tumour suppressor gene products act as a stop signal to uncontrolled growth, may inhibit the cell cycle or trigger apoptosis.
- In cancer, pick up mutations that are switch the genes off.
- Both genes for tumour suppressor must be mutated.
• TUMOUR SUPPRESSOR GENE = “Loss of function”
Describe how sarcoma virus was discovered.
- Chicken with sarcoma in breast muscle
- Remove sarcoma and break into small chunks of tissues
- Grind up sarcoma with sand
- Collect filtrate that has passed through fine-pore filter
- Inject filtrate into young chicken
- 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
- 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)
Why were retroviruses important experimentally?
Retroviruses were important experimentally:
- technological advances
- funding
- improved tissue culture techniques
- the discovery of reverse transcriptase, RNA genome, replicates via DNA intermediate and that they are enveloped.
What is the fundamental principle of oncogenes?
- Oncogenes are alerted forms of normal genes or proto-oncogenes
- c-src, cellular oncogenes
- v-src, proto-oncogene altered form transduced by retroviruses
Describe capture of c-src by retrovirus
- 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
1) infection and reverse transcription leads to dsDNA provirus
2) accidental integration of provirus next to c-src of host cell chromosomal DNA.
3) Co-transcription of viral (v-src) and c-src sequences
4) This is packages into capsids and leads to the formation of RSV virion carrying src sequences
What is the oncogene hypothesis?
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.
Describe viral oncogenesis.
Approximately 15%-20% of all human cancers are caused by oncoviruses
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
- 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
• 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.
What are some ways to activate an oncogene?
- Mutations
- Amplifications/duplications
- Translocation
- Leads to the loss of response to growth regulatory factors. Only ONE allele needs to be altered.
What are the four types of proteins involved in the transduction of growth signals?
NORMALLY:
- Growth factors
- Growth factor receptors
- Intracellular signal transducers
- Nuclear transcription factors
How does transduction of growth signals relate to cancer?
- 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
Describe the 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
- Most commonly mutated oncogene
- Point mutations in codons 12, 13 and 61
-Mutations leads to the loss of GTPase activity of the RAS protein normally required to return active RAS to the inactive RAS GDP.
=> Converts:
- Glycine to valine ~ bladder carcinoma
- Glycine to cysteine ~ lung cancer
Describe the normal mechanism of the RAS oncogene
- 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 factors to promote expression of genes
important for growth and survival
- Ras hydrolyzes GTP to GDP fairly quickly
Describe the MYC oncogene family.
- 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
- 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
Describe the MYC oncogene family (specifically it’s mutated role in cancer)
- 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
Describe 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. 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
Describe a case where chromosomal translocation is responsible for the activation of an oncogene
- 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
Describe retinoblastomas
The first tumour suppressor gene was identified by studies of retinoblastoma. 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%)
- Sporadic has to acquire 2 mutations and they develop retinoblastoma later on compared to those who have it inherited ~ Cell needs to be around long enough to get the second hit
The hereditary mutation is on chromosome 13 (13q14),
the retinoblastoma 1 (Rb1) gene
Give examples of different tumour suppressor genes
- regulators of cell cycle checkpoints (e.g. RB1),
- differentiation (e.g. APC)
- DNA repair (e.g. BRCA1)
Describe the retinoblastoma protein Rb structure.
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
What is the role of the retinoblastoma protein RB in the cell cycle?
- 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
Describe the link between RB function and phosphorylation.
- 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
Describe the inactivation of RB.
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 ~ E1A
- Papilloma ~ E7
- 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
What is p53?
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
Describe the structure of 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
How does MDM2 regulate p53?
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
How are p53 proteins activated?
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
Describe how targeting p53 is an efficient therapeutic strategy.
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
List some therapeutic strategies using p53.
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
Example of the use of inhibitors: - PRIMA-1, Restores mutant p53 by modifying the thiol groups in the core domain of the protein - Nutlin- is a potent MDM2 antagonist - RITA binds to p53 and can restore mutp53 activity - Inhibitors of CRM1 result in nuclear accumulation of p53
