Lecture 32 - Cancer Signalling Flashcards
Describe the trends in Australia in cancer
- Increased incidence
- Decreased mortality
- Increased rates of 5-year relative survival
Which signalling pathways are invariably altered in cancer?
- Cell survival
- Cell growth
- Cell differentiation
- Metastatic potential (?)
What is meant by signalling?
Biological communication at the submolecular / molecular level
Describe the Hanahan and Weinberg paper
Hallmarks of Cancer:
2000: • Evasion of apoptosis • Sustained angiogenesis • Immortal: limitless potential for replication • Metastasis and tissue invasion • Insensitivity to antigrowth signals • Self-sufficiency in growth signals
2011: • Avoidance of immune destruction • Tumour promoting inflammation • Genome instability • Deregulation of cellular energetics
Describe the major molecular events involved in cancer evolution
- Triggers / build up of events:
a. Environmental agents that damage DNA
• Chemicals
• Radiation
• Viruses
b. Inherited mutations in genes affecting:
• DNA repair
• Cell growth
• Apoptosis
- DNA damage
- Mutations in somatic cells
4.
• Impaired apoptosis
• Activation of oncogenes
• Inactivation of tumour suppressor genes
- Altered gene products
Abnormal structural and regulatory proteins - Malignant tumour
What is dysplasia?
Change in cell or tissue phenotype
Abnormality of development
Epithelial anomaly of growth and differentiation
Which classes of normal regulatory genes are the principal targets of genetic damage in carcinogenesis?
Characterise their involvement in carcinogenesis
- DNA repair genes
- Proto-oncogenes
- Tumour suppressor genes
- Genes that regulate apoptosis
In almost all cases of carcinogenesis, all classes are involved, and the pathways they are involved in interact
List the types of mutations that occur in cancer
- Errors in DNA replication that are not repaired
• e.g. BRCA1 & BRCA2
• Accumulation of errors in “hotspots” (TSFs, oncogenes) - Point mutations
• Activation of oncogenes
• Inactivation of TSGs - Amplification of oncogenes
• CNV - Chromosomal rearrangements
Mutation in which class of genes may often be an early event that allows rapid accumulation of secondary mutations?
Mutations in DNA repair genes
What is meant by mutation?
Change in a DNA sequence away from normal
This implies there is a normal allele that is prevalent in the population and the mutation changes this to a rare and abnormal variant
Describe how chromosomal translocations can lead to carcinogenesis
- Translocation between Chromosomes 9 and 22
- Fusion of the ABL and BCR loci
ABL: oncogene (tyrosine kinase)
BCR: Breakpoint cluster region
- BCR-ABL fusion protein
Constitutively active tyrosine kinase - Chronic myeloid leukaemia
What can immunohistochemistry tell us about cancer?
Fluorescently tagged mAbs that allow detection of markers of proliferation
Examples:
• PCNA: proliferating cell nuclear antigen
• Ki-67: recognises specific antigen in Hodgkin lymphoma
In tumour tissue there are very high levels of these markers of proliferation in comparison to normal tissue
Describe the rate neoplastic growth
Theoretically:
• 90 days to do from one cell to 10^9 (1g)
• After one more month, the tumour would weigh 1kg
• This would require all cells to remain in cell cycle and no cell loss
Realistically:
• Takes longer, because not all cell divide and some die
Lag phase: • Slow growth • Cells sensitive to the micro-environment • Immune regulated killing of cells • Poor blood supply
Growth phase:
• Exponential
• Angiogenesis: tumour has a rich blood supply delivering nutrients that allow it to rapidly increase in size
Plateau:
• Necrosis, because tumour is too big
• Growth slows
Describe the normal steps in proliferation
Which aspects of this could lead to carcinogenesis, to a greater and lesser extent?
- GF
- GF receptor
- Intracellular kinase
- Transcription; translation
- Cell enters cell cycle
- Proliferation
Carcinogenesis: Commonly: • Mutant receptor - always "on" • Mutant intracellular kinase • Mutant transcription factor
Not so common:
• Mutant cyclins and CDKs
• Over-expression of GFs
What is the name of the product of oncogenes?
Oncoproteins
Compare proto-oncogenes and oncogenes
Proto-oncogenes:
• Normall cellular genes
• Products almost always promote proliferation and suppress differentiation
Oncogenes:
• Mutant versions of proto-oncogenes
• Function autonomously without requirement for normal growth-promoting signals
Which transduction pathway is normally activated with GF signalling?
- MAPK
* PI3 kinase
What type of receptor are GF receptors?
Tyrosine kinase receptors
Mutations in how many alleles is required for carcinogenesis in oncogenes and TSGs?
Compare the outcome of mutation in oncogenes and tumour suppressor genes
Oncogene mutation:
• Only 1 allele mutated
• Accelerated growth
Tumour suppressor gene mutation:
• Both alleles lost
• Continuous growth
Give examples of oncogenes and TSGs
Oncogenes:
• Her2-neu
• Ras
• Myc
TSGs: • p53 • Rb • APC • PTEN
What do TSGs usually encode?
Proteins that inhibit cellular proliferation by regulating the cell cycle directly:
• Rb
• p53
• PTEN (inhibition of oncogenic pathways)
Describe LOH
Loss of heterozygosity
Part of Knudson’s “two hit” hypothesis
- One allele for the given TSG is already mutated (heterozygosity)
- Loss of normal function of the other allele
- LOH
(Both alleles of TSGs must be lost for tumour development)
Describe Knudson’s “Two Hit” hypothesis
Loss in one allele of a TSG is not enough to generate a tumour
LOH needed to form the tumour
Initial ‘hit’: i.e. loss of TSG may be:
• Inherited: earlier onset
• Sporadic
Describe the different ways that functional TSGs can be lost
– Deletion –
- miRNA –
1. TSG transcribed into mRNA and leaves nucleus
2. Abundant miRNA in cytosol, binds to target TSG mRNA
3. TSG mRNA not transcribed
4. Reduced tumour suppressor protein
- miRNA –
- Epigenetic changes –
1. Focal CpG hypermethylation of promoter or start of transcription site of TSG
2. TSG not expressed
- Epigenetic changes –
What are miRNAs?
- Non-coding
- Single stranded RNA
- approx. 22 nucleotides
Function:
• Negative regulators of genes
• Through binding mRNA and preventing translation
What is the mechanism through which cell division is controlled?
Describe its importance in cancer
Cell cycle
“Cancer is a disease of the cell cycle”
Since:
• Cancer is uncontrolled division
• Division controlled by cell cycle
Which important functions does the cell cycle perform?
- Regulation of the growth and mitotic phases
* Ensures faithful replication and segregation of genetic material
What is the effect of oncogenes and TSGs on the cell cycle in cancer?
Cell cycle checkpoints are perturbed
Checkpoints:
• Checking the integrity of the DNA at various points in the cell cycle
Both TSGs and Oncogenes are involved in the checkpoints
TSGs:
• Normally stall the cell cycle
• e.g. p53
Oncogenes:
• Cause acceleration / bypass through the checkpoints
Describe the role of p53 in carcinogenesis
p53:
• TF
• Regulates expression of cell cycle factors
Function
1. Cellular stress
- Up-regulation of p53
- Transcription of tumour suppressing genes
4. Cellular outcomes • Apoptosis • Cell-cycle arrest • DNA repair • Differentiation • Senescence
In cancer:
• Involved almost invariably
• Loss of p53 → cells no longer able to conduct normal regulatory functions (DNA repair, apoptosis etc)
Describe evasion of apoptosis in carcinogenesis
Apoptosis must be avoided for tumourigenesis
Mechanisms:
• Reduced CD95 (aka FAS)
- Inactivation of death-induced signalling complex (aka pro-caspase 8) by FLICE
- Upregulation of BCL-2
- Reduced levels of BAX
- Loss of APAF-1
- Up-regulation of inhibitors of apoptosis
Describe how the FAS-FASL pathway can be interrupted leading to tumour generation
- Reduced FAS expression on cell
- FLICE protein
• Inactivates the death-induced signalling complex (aka pro-caspase 8)
Describe the role of BCL-2 in tumour generation
BCL-2 is an anti-apoptotic
In cancer it can be up-regulated
Describe the role of BAX in tumour generation
BAX is a pro-apoptotic
Loss of p53 → decreased BAX
What is the role of APAF-1 in cancer?
APAF-1 is a pro-apoptotic
APAF-1 activates caspase 9 → apoptosis
Loss of APAF-1 → tumour growth
Describe how tumour cells can become immortal
Telomeres endow limitless replicative potential (→ cancer)
Normal role of telomeres:
- Telomerase adds more nucleotides to the telomeres so that they don’t become too short and thus lose integrity
- Normally low telomerase activity in cells
- Shortened telomeres eventually activate cell cycle checkpoints → senescence
In cancer:
• Shortened telomeres due to many cell replications
- Reactivation of telomerase
- Cells become immortal (avoid senescence)
Describe generation of metastasis and the importance in carcinogenesis
Metastasis is important for tumour growth
Mechanism:
1. Detachment of tumour cells from one another
• Down-regulation of adherence molecules
• E-cadherin
• Beta-catenin
• Integrins
- Degradation of ECM
• Metalloproteinases expressed at high levels - Attachment to novel ECM components
- Migration in the blood
Describe the importance of angiogenesis in carcinogenesis
Angiogenesis is important for the support of tumour growth.
They are very rapidly growing, and thus require a rich blood supply
Mechanism:
• VEGFs and VEGFRs upregulated
• Increased recruitment of vascular stem cells
• Angiogenesis
This is a popular therapeutic target
“Anti-angiogenics”
Describe tumour cell heterogeneity
What is this due to?
Tumours are very heterogeneous
Even in one tumour, the genomes of cells in one region can be very different to the genomes of cells in another region
Tumour cells have the ability to mutate rapidly
Due to:
• Genomic instability
• Through loss of p53
• Loss of DNA repair proteins
This is probably why tumours rapidly become resistant to drugs
What can tumour initiating cells do?
Allow human tumour growth when transplanted into immunodeficient mice
Describe cancer stem cells
Have a high intrinsic resistance to conventional therapies
Make up only a small part of the bulk of the tumour
Describe how cancer stem cells could be targeted, and how they are conventionally targeted
Cell mass containing cancer stem cells and tumour cells
Conventional therapy:
• Targets bulk of the tumour cells and not cancer stem cells
• Tumour relapse from cancer stem cell
Cancer stem cell specific therapy:
• Targets cancer stem cell and not cells that form the bulk of the tumour
• Tumour regression
Describe how the various aspects of Hanahan & Weinberg’s model of cancer can be therapeutically targeted
- Self-sufficient growth factor signalling
• EGFR inhibitors - Evasion of growth suppressors
• CDK inhibitors - Avoidance of immune destruction
• Immune activating anti-CTLA4 mAb - Immortality
• Telomerase inhibitors - Angiogenesis
• Inhibitors of VEGF signalling - Metastasis
• Inhibitors of HGF/c-Met - Genome instability and mutation
• PARP inhibitors - Deregulation of cellular energetics
• Aerobic glycolysis inhibitors - Resistance of apoptosis
• Pro-apoptotic BH3 mimetics - Tumour promoting inflammation
• Selective anti-inflammatory drugs
Discuss biochemical ‘circuits’ and their role in cancer and the therapeutics thereof
Complex interaction between various signalling pathways that modulate the outcomes
Can be divided into very specific functions
Examples: • Proliferation circuits • Viability circuits • Cytostasis and differentiation circuits • Motility circuits
Implication for therapeutics:
• This is important to consider when targeting cancers with therapeutics
- Targeting a single factor in cancer works for a few months, but is incapable of continual inhibition
- The tumour will evolve resistance to this particular drug
- Drugs must target multiple signalling centres within a tumour
What proportion of cancers have an inherited component?
Only 15%
Thus, most cancers are stochastic (environmentally related)
What are the functions of the BCRA1 and BRCA2 genes?
How does this lead to tumour growth?
They are DNA repair genes
If there are mutations in these genes, DNA can not be repaired and there will be an accumulation of errors
(often in proto-oncogenes and TSGs)
Can lead to Breast, Ovarian and Pancreatic cancer
What are some important DNA repair genes whose mutations are involved in cancer?
Describe their function
- BCRA1
- BCRA2
- MSH2
- MLH1
In the process of DNA replication, there will inevitably be mistakes and damage to the DNA
There are specific genes that encode proteins that repair DNA when this happens
Mutations in these genes can lead to an accumulation of other mutations (e.g. in proto-oncogenes, TSGs etc)
What damage can occur to DNA during replication?
- Single stranded breaks
- Double stranded breaks
- Bulky adducts
- Mismatches
- Insertions
- Deletions
- Methylation
Describe mutations causing neuroblastoma
Massive amplification of a region on the chromosome that encodes n-myc (an oncogene)
N-myc regions also break off and form “Double minutes”: look like tiny chromosomes
“Double minutes” also able to form the n-myc proteins and drive the malignancy
What is the role of differentiation in cancer?
Suppression of differentiation
Cells remain in an immature state
Immature cells continuously grow and divide
Thus, suppression of differentiation drives tumourigenesis
What is the role of PTEN?
What about in cancer?
- PTEN is a TSG: blocks oncogenic pathways
- Normally blocks the PI3 kinase transduction pathway
Mutations:
• No longer able to inhibit PI3 kinase pathway
• Cell proliferation and growth
What sort of mutation in the PI3 kinase pathway would lead to tumourigenesis?
Mutation so that the pathway is constitutively active
Describe the interaction of PI3 kinase and PTEN in cancer
Pik3ca: oncogenic form of PI3 kinase gene
PTEN: suppressor of PI3K
Ovaries with various mutations:
- WT: normal sized ovaries
- Pik3ca mutation: not much difference
- PTEN KO: not much difference
- Pik3ca;PtenKO: massive tumours
Brain; lateral ventricles (normally contain many stem cells)
• WT: no tumours
• Mutant (Pik3ca;PTEN KO): massive tumours growing into lateral ventricles
Implication:
• Need oncogenic mutations as well as loss of Tumour suppression
What is special about Retinoblastoma?
Cancer of the retina
Only cancer that is dependent on only one gene (Rb)
Describe the cell cycle
G1: growth G1/S checkpoint: checking DNA S: DNA replication G2: growth phase G2/M checkpoint: checking of DNA integrity after replication M: cell division
Describe the various mechanisms of apoptosis
A. Extrinsic
- FasL-Fas interaction
- FADD activation
- Activation of caspases
- Cell death
B. Intrinsic:
- Environmental stress (radiation, chemicals)
- DNA damage
- p53 response
- BAX
- Mitochondria loss of cytochrome c
- Activation of caspases
- Apoptosis
What is the role of E-cadherin in cancer?
Holds cells together
Loss of E-cadherin allows cells to metastasise
Which hallmark of cancer has been the target of a large proportion of therapeutics?
Angiogenesis
