43. Neoplasia: The Molecular & Cellular Basis Of Tumour Growth (HT) Flashcards
Give some experimental evidence relating to the two-hit hypothesis of cancer.
[EXTRA]
(Knudson, 1971):
- Studied familial retinoblastoma
- Concluded that both alleles of a tumour suppressor locus must be mutated in order for a tumour to form
Give some experimental evidence relating to oncogenes and tumour supressor genes.
[EXTRA]
- In 1976, Harold E. Varmus and J. Michael Bishop discovered the first cellular oncogene, called src.
- In 1986, Stephen H. Friend et al. isolated the first tumor suppressor gene, called Rb (short for retinoblastoma)
Give some experimental evidence relating to DNA microarray chips.
[EXTRA]
- A DNA microarray contains several DNA spots on a surface.
- These microarrays are used to measure the expression levels of large numbers of genes simultaneously or to genotype parts of a genome.
- In 1995, the first DNA microarray chip was constructed.
- This has enabled individualised treatment of cancer based on the underlying changes.
Give some experimental evidence relating to creation of tumour cells.
[EXTRA]
(Hahn WC et al., 1999):
- Managed the first successful creation of tumor cells
- Human epithelial and fibroblast cells were transformed into tumour cells.
- This involved the co-expression of only 3 genes, which is unusually few compared to in vitro cancers: telomerase (hTERT), simian virus 40 large-T oncoprotein, and an oncogenic allele of H-ras
What are the original six “hallmarks of cancer”? Who came up with them?
[EXTRA?]
Douglas Hanahan and Robert Weinberg (2000)
For each of the original six “hallmarks of cancer”, give an example of how this hallmark may arise.
Describe how the original six hallmarks of cancer were later expanded.
Douglas Hanahan and Robert Weinberg (2011):
- Added two new hallmarks
- Also added two new enabling characteristics which can help the development and spread of cancer
Describe the cells that are in the micro-environment of a tumour.
- Cancer cells
- Cancer stem cells
- Cancer-associated fibroblasts
- Endothelial cells
- Pericytes
- Immune inflammatory cells
- Invasive cancer cells
It is worth noting that the types of cells seen in a metastatic tumour environment will be different than those seen in a different tissue.
As a general principle, what underlies the growth of tumours?
Signalling between the different cells in the micro environment.
What tumour cells are responsible for metastasis?
Cancer stem cells, since they have a high potential for renewal and proliferation, so they can lead to the formation of new tumours.
Can a tumour contain more than one tissue type?
Yes, because the cancer stem cells can be multipotent, which means that they can differentiate into various cell types.
Describe an experiment that gives evidence for the existence of cancer stem cells.
[EXTRA]
- Tumour cells from a mouse are purified
- The cells are injected into an immunodeficient mouse, where another tumour develops
- This process is repeated with another mouse
- This demonstrates that there must be some cancer stem cells that allow this continued renewal and proliferation
Summarise the main categories of curative treatments for cancers and state how common each is.
What are the main categories of cytotoxic drugs used to treat cancers?
- Antimetabolites
- Alkylating agents and platinum drugs
- Topoisomerase inhibitors (a.k.a. anti-tumour antibiotics)
- Anti-mitotic agents (a.k.a. microtubule poisons)
What are the general modes of action of the main types of cytotoxic drugs used to treat cancers?
They inhibit cell proliferation and induce cell death.
What factors does the growth rate of a tumour depend on?
- Growth fraction (percentage of proliferating cells within a given system)
- Cell cycle time
- Rate of cell loss
According to the spec, what factors limit the growth rate of a tumour?
[IMPORTANT]
- Many cells are not actively proliferating
- Cellular differentiation
- Death (necrosis or apoptosis)
- Cell loss (e.g. from skin and gut, or shedding into the circulation).
What fraction of cells in a tumour must be killed in order to eliminate that tumour?
Almost 100%, because tumour cells proliferate roughly every 24 hours, so the tumour will grow back to its original size very quickly unless just about all of the cells are killed.
What chemotherapy strategy may be used to eliminate a tumour without excessive toxic effects?
3 logs kill, 1 log re-growth:
- Chemotherapy is used to reduce the number of cells by a power of 103 (e.g. from 1012 to 109 cells)
- The cell number is then allowed to increase by a power of 101, which allows the patient to recover from the toxic effects
- This is then repeated until the tumour is eliminated
Summarise the different aims of chemotherapy.
- Curative
- Neoadjuvant -> When the chemotherapy is before surgery
- Adjuvant -> When the chemotherapy is after surgery
- Palliative
- Reduces tumour bulk
- Slows the growth of existing lesions
- Delays development of new lesions
- Relieves symptoms
Name some tumour types that have high, medium and low sensitivity to chemotherapy.
[EXTRA]
How do antimetabolite drugs work as cytotoxic drugs to treat cancer? Give an example.
[IMPORTANT]
- They metabolically inhibit DNA synthesis
- Example: Methotrexate
How does methotrexate work?
It is an anti-metabolite:
- It is a competitive inhibitor of dihydrofolate reductase (DHFR)
- This inhibits synthesis of purines and dTMP
- Thus, this inhibits RNA synthesis and DNA replication
This leads to slowing of the growth of the tumour, as well as in other diseases (e.g. in rheumatoid arthritis).
Give an example of an antimetabolite with a similar action to methotrexate.
5-fluorouracil (5-FU) is converted in the body to FdUMP, which inhibits thymidylate sythase (TS).
What are some examples of DNA damage-based therapies for cancer and what their effect is?
These all work by inducing double-strand breaks.
How do alkylating agent drugs work as cytotoxic drugs to treat cancer? Give an example.
[IMPORTANT]
- They chemically damage DNA.
- Examples: Cyclophosphamide, Cisplatin
How does cyclophosphamide work?
It is an alkylating agent:
- In vivo it is oxidised to phosphoramide mustard
- The chlorines can take part in displacement reaction, which cross-link the DNA strands and can induce damage
It is effective in treating cancer and other conditions (e.g. rheumatoid arthritis)
What determines the selectivity of cyclophosphamide?
- The aldophosphamide intermediate is detoxified by aldehyde dehydrogenase (ALDH)
- This confers some selectivity for cells with low ALDH
How does cisplatin work?
It is an alkylating agent:
- Displacement of the chlorine allows cross-linking of the DNA strands
- This leads to cytotoxicity
It is effective in treating cancer.
How was cisplatin discovered and by who?
[EXTRA]
(Rosenberg, 1965):
Discovered by chance observation of anti-bacterial properties of platinum electrolyte.
What cancers is cisplatin especially good at treating?
[EXTRA]
- It is very effective in testicular cancer, even if there is metastasis.
- It led toa rise in cure rate from 10% to 80%.
What category of drugs do topoisomerase inhibitors belong to?
Anti-tumour antibiotics
How do topoisomerase inhibitors work as cytotoxic drugs to treat cancer? Give an example.
[IMPORTANT]
- They bind to DNA and can lead to double-strand breaks.
- Examples: Doxorubicin
How does doxorubicin work?
It is a topoisomerase inhibitor (a type of anti-tumour antibiotic):
- It inhibits DNA topoisomerase II, which is involved in mitosis
- This inhibition essentially leaves both DNA strands broken (i.e. causing double-strand breaks)
It is effective in treating cancer.
What determines doxorubicin’s selectivity?
[EXTRA]
- Topoisomerase is expressed most during G2 of the cell cycle.
- This means that the cells in this phase are most susceptible to doxorubicin.
How do anti-mitotic agents work as cytotoxic drugs to treat cancer? Give an example.
[IMPORTANT]
- They inhibit mitosis by blocking microtubule action
- Example: Vinca alkaloid (e.g. vincristine)
How does vincristine work?
It is a microtubule poison:
- Inhibits tubulin polymerisation
- This prevents the spindle assembling properly during mitosis
- This causes the cell to be unable to progress past metaphase and it dies by apoptosis at the next mitotic checkpoint.
It is effective in treating cancer.
What is the danger of vinca alkaloids (like vincristine)?
- They are severely neurotoxic, since they inhibit microtubules, which are essential in neurons for neurotransmitter transport.
- Therefore they cannot be administered into the CSF, but must be administered via IV.
Summarise the side effects of chemotherapy.
What are the two types of side effects of cytotoxic chemotherapies for cancer?
[IMPORTANT]
- Reversible cytotoxic effects (e.g. damage to bone marrow, lymphoid tissue, GI epithelium, hair)
- Irreversible toxicity to organs with little/no cell growth (kidney, nerves, heart & lungs).
What is the problem of the side effects of chemotherapy?
They limit the concentrations of chemotherapy drugs that can be used.
Which organ is most dangerously affected by cytotoxic chemotherapy drugs? Why?
Bone marrow:
- Destruction of the bone marrow can lead to low neutrophil counts (and low levels of other cells)
- This makes the individual susceptible to infections
- A neutrophil count of <0.5 x109/L with fever is strongly predictive of bacteraemia (neutropenic sepsis)
- Thrombocytopenia and anemia also common
How can the effects of cytotoxic chemotherapy on bone marrow be protected against?
- In the case of infection, IV / oral antibiotics are essential
- Recombinant G-CSF is used prophylactically to boost neutrophil levels
Describe the effects of cytotoxic chemotherapy on alopecia.
- Occurs from 2 days to a few weeks after therapy
- Hair returns around 3-6 months after stopping treatment
- A cold cap can be used to reduce blood flow to the hair, so that hair loss is limited
Describe the concept of extraversion of cytotoxic chemotherapy drugs and how it can be reduced.
[EXTRA?]
- When chemotherapy drugs are administered via IV, the drug may leak from the bloodstream into surrounding tissue due to poor insertion of the canula or due to weakness of the blood vessel
- This leads to severe damage of surrounding tissue
- Therefore, a peripherally-inserted central catheter (PICC) can be used to deliver the drug straight to the vena cava, so that it is very rapidly diluted -> This reduces the risk of damage upon extraversion
Describe hand and foot syndrome.
- A skin reaction around 2-12 days after chemotherapy
- It invovles vessel damage in the hands and feet
- Symptoms incude tingling, burning pain, peeling
- Resolution occurs 1 to 2 weeks after stopping treatment
Why are combination chemotherapies used?
- Staggered schedule and/or synergy can increase efficacy/reduce toxicity
- Minimises the likelihood of developing resistance
What is therapy-induced tumorigenesis?
- The idea that most cytotoxic therapies for cancers are also carcinogenic
- This can lead to new tumours forming that are distinct from the original disease
- Around 1 in 6 of all new cancer diagnoses in the UK are due to this
What are some other types of treatments for cancer aside from cytotoxic drugs? Why are they used?
[IMPORTANT]
- Hormonal therapies
- Molecularly targeted therapies
They are used because cytotoxic therapies are very toxic and lead to severe side effects.
Give some examples of hormonal drugs for treatment of cancers.
- Tamoxifen
- Aromatase inhibitors
- Orchidectomy
- Drugs to depress LH release
- Anti-androgens
(These are the ones mentioned in the spec)
How does tamoxifen work?
It is a hormonal therapy for breast cancer:
- It is metabolised to hydroxytamoxifen
- This is an estrogen receptor (ER) antagonist
- Thus, it stops the growth of breast cells
- It may also have other unknown mechanisms of action
What is a side effect of tamoxifen?
It has estrogenic (agonist) effects in endometrium, which is opposite to the effect it has on the breasts.
How do aromatase inhibitors work?
They are a hormonal therapy for breast cancer:
- Block peripheral estrogen synthesis in post-menopausal women
- Used in breast cancer
Name two types of inhibitors of growth signal transduction.
- Therapeutic antibodies against growth factor receptors (e.g. trastuzumab)
- Small molecule inhibitors of cell cycle enzymes (e.g. imatinib)
Summarise the main checkpoints in the cell cycle.
Summarise the main drugs that act at each of these cell cycle checkpoints.
Summarise the main mechanisms by which resistance to chemotherapy drugs for cancer can occur.
[EXTRA]
- Decreased drug entry into cells
- Altered expression or mutation of target enzymes
- Improved DNA repair activity
- Decreased tendency to apoptosis
- Improved detoxification
- Drug extrusion (multi-drug resistance)
Give an example of a condition that predisposes to cancers and the mechanism by which this happens.
[EXTRA]
What are oncoviruses often accompanied by and why?
- The viral genome is small and therefore when an oncogene is inserted, it often displaces important genes
- This means that the virus may be helped by a helper virus that encodes components of the virus, such as gag, pol and env
- Together, they are selected for because they increase cell proliferation and thus the rate of generation of new viruses
What hallmark of cancer do oncogenes usually affect?
- They mostly cause increased proliferative signalling (or evasion of growth suppressors) -> These lead to uncontrolled growth and proliferation
- They can also have other effects, such as reducing apoptosis, which contribute to tumour development
(Check this, but it seems that oncogenes must cause increased proliferation, but can also have other effects on the hallmarks of cancer)
Describe how oncogenes can lead to increased proliferation of cells.
[EXTRA?]
The oncogenes can encode mutated proteins at various points along the proliferative pathways. For example, in receptor tyrosine kinase pathways there may be:
- Mutated growth factors (e.g. v-sis oncogene)
- Mutated growth factor receptor (e.g. v-erbB oncogene)
- Mutated RAS protein, so that it is more likely to bind GTP (e.g. v-ras oncogene)
- Mutated RAF protein (e.g. v-raf oncogene)
Give some examples of dominant mutations (i.e. oncogenes) in human cancers.
Give some experimental evidence for the use of genome studies to identify cancer-relevant mutations.
[EXTRA]
(Davies, 2002):
- Sequencing studies of tumours of malignant melanoma found a single nucleotide mutation in 60% of tumours
- This mutation involved a single nucleotide change on one of the chromosomes encoding the BRAF protein (shown in the diagram)
- Thus, this mutation could cause cancers by stimulating the receptor tyrosine kinase pathways shown
(Patton, 2005):
- This was demonstrated by use of animal models
- Zebrafish with this BRAF V600E mutation demonstrated that this protein induced a high rate of development of melanomas in the fish compared to controls
(Chapman, 2011):
- Deduction of the structure enabled the creation of an inhibitor called vemurafenib (BRAF inhibitor)
- This induced clinical responses in 77% of metastatic melanoma patients with the BRAF V600E mutation
How can replicative immortality of cells occur?
[EXTRA]
Mutations in the promoter region of the telomerase reverse transcriptase (TERT) gene, which encodes part of telomerase, can lead to increased telomerase activity that maintains the cell’s lifespan.
However, note that telomerase is not strictly a proto-oncogene since it does not lead to increased cell proliferation!
Describe Knudson’s two hit hypothesis.
- Both copies of a tumour suppressor gene must be mutated in order for a cancer to develop
- This means that familial pre-dispositions to cancer arise when one of the alleles encoding the tumour suppressor gene is already mutated, so that only one more must be mutated in order to cause cancer
NOTE however, that both tumour suppressor alleles being mutated is not usually sufficient for the development of a malignant tumour. Other mutations must happen in addition to this, as per the hallmarks of cancer.
Describe how tumour suppressor genes may be identified experimentally.
[EXTRA]
- Linkage and positional cloning involve identifying the approximate locus of the tumour suppressor gene by studying which polymorphic markers it is most frequently inherited with.
- Association of gene product with DNA viral oncoprotein involves looking at which host cell proteins are affected by oncoviruses, meaning that these proteins may be tumour suppressor proteins.
- Cancer genome projects that use NGS have accelerated the speed at which tumour suppressor genes are found.
Give some examples of loss-of-function mutations in human cancers.
[EXTRA]
Describe how tumour suppressor gene mutations (and oncogenes) may lead to sustained proliferative signalling and evasion of growth suppressors.
[EXTRA?]
The restriction point (R) is the point in G1 that marks commitment such that the cell no longer needs growth factors to finish the cell cycle. It may be affected by tumour suppressor gene mutations:
- RB inhibits the E2F-DP transcription factor complex that leads to transcription of genes that are needed for S phase -> RB mutations can predispose to familial retinoblastoma and various sporadic cancers
- Cyclin D is a CDK that phosphorylates the RB, inhibiting the inhibition of the transcription factors -> Cyclin D may be overexpressed due to gene amplification and oncogene signalling, which predispose to cancers (so this is really a proto-oncogene)
- CDK inhibitors inhibit the action of cyclin D -> p16 mutation / promoter methylation, increase p27 degradation and CDK4 mutation (causing dysfunctional interaction with p16) may all predispose to cancers
Describe how tumour suppressor gene mutations may induce angiogenesis.
[EXTRA?]
For example, tumour suppressor gene mutations may affect the HIF pathway:
- In hypoxia, HIF-alpha leads to transcription of various genes including VEGF
- This VEGF leads to increased angiogenesis
- VHL is part of the complex that degrades HIF upon return to normoxia -> Mutations in VHL lead to sustained angiogenesis (VHL is a tumour suppressor gene)
Describe how tumour suppressor gene mutations may lead to resisting of cell death and evasion of growth suppressors.
[EXTRA?]
For example, p53 tumour suppressor gene mutations:
- p53 is a tumour suppressor gene responsible for inducing cell cycle arrest, apoptosis or differentiation in cells affected by DNA damage, hypoxia and oncogene activation:
- p53 stimulates inhibitors of CDK, such as p21 and 14-3-3-sigma -> This halts the cell cycle
- p53 stimulates BAX, noxa and PUMA, which lead to release of cytochrome c from mitochondria. p53 also drives the expression of death receptors. -> This leads to apoptosis
- p53 mutations therefore predispose to various types of cancers
Are p53 mutations found in all cancers?
[EXTRA]
- TP53 (the gene encoding p53) mutations are found is very many cancers
- However, in some cancer types, a lack of p53 mutation may be compensated for by a different mutation
- For example, renal cancers very commonly have VHL mutations instead of p53 mutations
Describe how p53 is activated by DNA damage and oncogenes.
[EXTRA]
- p53 is usually unstable because one of its effects is MDM2 transcription, which feeds back and degrades the p53
- However, ATM (increased by DNA damage) and ARF (increased by oncogene activation) inhibit this feedback degradation
- This means there is more p53 available to carry out apoptosis, etc.
Can p53 (tumour suppressor) function be lost only by mutation of the p53 protein?
[EXTRA]
No, p53 dysfunction (leading to predisposition to cancer) may also be caused by:
- MDM2 overexpression
- Loss of ATM function
- Viral infection
- Loss of ARF -> Involving deletion or promoter methylation of the CDKN2A gene that codes for ARF and p16
Are p53 mutations always loss of function mutations?
[EXTRA]
- Often they are, which means that there is a lack of p53 function and thus there is reduced apoptosis, etc.
- However, sometimes mutations in regions of the p53 protein other than the DNA-binding domain may be gain of function mutations
- These gain of function mutations can ‘poison’ other p53 molecules in a tetramer, inactivating them -> This is actually a dominant effect, which means that even one of these mutations can leads to a significant reduction in apoptosis, etc., even if the other allele is still unaffected
Summarise conceptually how a tumour develops and then metastasises.
- Normal tissue (e.g. epithelial) has excess growth, forming a primary tumour
- There is also angiogenesis and secretion of proteases (by tumour cells or stromal cells) that destroy the tissue structure
- When there is loss of contact between cells, there is deattachment of cells
- If the basement membrane at the boundary of the organ is affected by proteases, this releases cells from the tumour
- The cells invade lymphatic and blood vessels
- The cells are deposited at distant sites that can remain dormant for long periods of time, but they are be reactivated by a similar process to the initial tumour, leading to new tumours
Summarise the main steps required for metastasis to occur.
[EXTRA]
- At the top, there are the steps needed for a primary tumour to grow and then the environmental changes required for it to spread to vessels.
- In the middle, there are the steps involved in transit to a metastatic site.
- At the bottom, there are the steps involved in invasion developing a metastatic tumour.
Note that not all of these steps are required always and they may not happen in this exact order.
Give an example of how microenvironmental changes in the primary tumour can lead to metastasis.
[EXTRA?]
- Microenvironmental changes are contributed to by two-way interactions between tumour cells and stromal cells, such as macrophages and fibroblasts.
- In experiments, cultured macrophages alone produce a small amount of MMP (matrix metalloprotease) and breast cancer cells alone produce very little MMP.
- However, when macrophages and breast cancer cells are combined, the MMP production is greatly increased.
- This is because the CSF on cancer cells activates macrophages’ MMP and EGF production
- Then EGF (a growth factor) on macrophages reciprocally stimulates proliferation
- High levels of MMP lead to degradation of the tissue structure, including basement membrane breakdown, etc. -> This causes release of cells that can enter the bloodstream
What important transition underlies intravasation (invasion of cancer cells through the basement membrane into a blood or lymphatic vessel) during metastasis? Why?
[EXTRA?]
- Epithelial-to-mesenchymal
- This is because epithelial cells have strong basolateral cell-to-cell adhesions that maintain the structure
- Without this, the cells are more free to cross the basement membrane into the vessel (i.e. greater migratory potential)
What changes occur in epithelial-to-mesenchymal transitions that enable greater migratory potential (relevant to metastasis)?
[EXTRA?]
- Loss of basolateral adhesions that maintain rigid structure -> So they can move more freely
Other changes:
- Cell becomes more elongated
- (Maintained/Increased?) Expression of focal cell-cell contacts that enable some adhesion to other cells but importantly also to the ECM -> So greater migratory potential
What underlies epithelial-to-mesenchymal transitions in metastasis?
[EXTRA?]
- The process is the same as in the creation of mesoderm (the 3rd germ layer) from the epithelial layers during gastrulation
- The mechanism involves a cascade that results in reduced expression of adhesion molecules (cadherin), which frees up the cells for migration
- The signalling pathway for this is retained later in life and chance alterations in expression (genetic or epigenetic) of some of the factors in these pathways leads to unwanted epithelial-to-mesenchymal transitions in cancers. This leads to metastasis.
- In carcinomas, the pathway is more complicated though (shown on the left), and also includes loss of desmosomes and changes to the cytoskeleton.
Name some of the transcription factors that control epithelial-to-mesenchymal transitions. How do they relate to cancer?
[EXTRA]
- These transcription factors control EMT during gastrulation and similar processes.
- However, they can also by chance become expressed in tumours, which causes the cells to be disorganised and separated from each other.
- This increases the likelihood of the cancer cells crossing the basement membrane into blood and lymph vessels.
Draw a diagram to summarise how exosomes released by tumour cells influence the location of future metastases.
- Exosomes with different integrins bind at different sites in the body
- They are taken up by cells and alter their function (e.g. by reprogramming energy metabolism), such that a pre-metastatic niche forms
- This increases how favourable the environment is for tumour formation, so that circulating tumour cells may enter there and start a tumour
Summarise some of the functions that tumour cell exosomes can exert on recipient cells.
Summarise how extravasation (exiting from the blood into tissue across the basement membrane) of circulating tumour cells can occur.
- When a circulating tumour cell reaches a capillary it is often too large to pass through
- This causes it to damage the endothelium, leading to recruitment of platelets and thrombus formation
- As the thrombus is lysed, there is also some damage to the endothelium, so that the tumour cell can attach to the basement membrane.
- Proliferation of the tumour cells leads to physical rupture of the basement membrane.
- MMP release by the tumour cells also aids this rupture.
Why are micro-metastases particularly dangerous?
- They are formed by tumour cells that have escaped a primary tumour, meaning that this property has been selected for.
- Therefore, most of the cells that arise from the metastatic tumour (created when the micro-metastasis grows) already have the properties for escaping the site.
- Thus there may be a cascade of new metastatic sites around the body.
What is the relationship between BRCA1 and metastasis?
[EXTRA]
- BRCA1 mutations not only increase the risk of developing breast cancer, but also increase the risk of metastasis
- This is because BRCA1 is thought to play a role in reducing the motility of cells
- Thus, increased rates of metastasis are seen in the years after recovery in BRCA1 mutation-carrying patients
- This is not only in breast cancers, but also in other cancers
- Identification of genes that slow the rate of metastasis is a promising concept in developing drugs that prevent metastasis
Name some therapeutic approaches to preventing metastasis.
[EXTRA]
