Cancer as an evolutionary process Flashcards
How cancer mutations affect regulatory pathways - Cell cycle control
a) normal control of the cell cycle
b) effect of cancer mutations
a) Cell cycle is divided into phases - G0 + G1 (diploid state), S (DNA synthesis), G2 (tetraploid state, completion and checks on S phase and preparing for M), and M (mitosis).
pRb protein (retinoblastoma protein) controls the cell cycle by inhibiting the inititation of DNA replication (halting the cycle at the G1/s checkpoint. The inhibition is relieved when Cyclin-Dependent Kinase 4 (which is activated by Cyclin D1 binding) phosphorylates pRb, inactivating it. The formation or activity of Cyclin D/CDK4 complex is inhibited by proteins, such as p16 (aka CDKN2A) and p21 (aka CDKN1A)
b) The brake (by pRb) can be taken off the cell cycle by mutating any of the components. In the case of pRb, it is inactivated (eg by deletion of all or part of the RB1 gene). In contrast, over-activating mutations of CDK4 or Cyclin D1 can persistently phosphorylate pRb (persistently inactivate). The CCND1/CyclinD1 gene is often overactive in breast cancer as a result of the cell having many extra copies of the gene (gene amplification). p16/INK4A inhibits formation of the active dimeric Cyclin D1/CDK4 kinase. It is often inactivated by deletion of both copies of the gene
a) ongogenes vs tumour suppressor genes
b) example of RB1 inactivation vs CDK4 or CyclinD1 activation
c) example of p53
a) An oncogene positively affects growth control, whilst a tumour suppressor gene negatively affects it. Oncogene mutations cause ‘over-activation’. Tumour suppressor gene mutations cause loss of function. Ongogene mutations are dominant in the cell (only one copy has to be mutated) while for tumour suppressor genes generally both copies have to be mutated, so the mutation is recessive in the cell.
b) for RB1, both gene copies have to be mutated because the cell can survive with only one functional copy making protein, making RB1 a ‘classic’ tumour suppressor gene. Oncogenes CDK4 or CyclinD1, which are activated by mutations, making just one copy overactive will suffice
c) p53 is an intermediate case. Some loss of function mutations (some tumour suppressor gene mutations) have a significant effect on the cell when only one copy of the gene is mutated, but losing both copies has a stronger effect. These are usually cases where the mutant protein forms complexes with the normal protein. p53 functions as a tetramer, so mutating one of the two copies of the gene means that most of the remaining WT protein is contained in the tetrameric complexes that are faulty. The presence of mutant protein in the tetramer compromises its function.
How tumours develop by Darwinian evolution
Cancers develop by successive evolution of clones of cells that have a selective advantage over their neighbours. A normal cell acquires a mutation/gene change that means that, over time, its progeny compete with neighbouring cells so that they take over more than their normal share of a tissue. This is clonal expansion. Then on eof these progeny acquired a further mutation that confers a selective advantage, and so on. At some point the growth pattern becomes visibly distorted.
Later the tumour becomes malignant and it will continue to become more aggressive and may develop resistance to therapy if treated. Therefore, the tumour progresses. A lump of tumour may contain not just the latest clone, but also preceding clones and dead-end branches of the evolutionary tree, so there may be some heterogeneity in the sample
Demonstration of clonal expansion of tumours
By Phil Jones and colleagues (see image). Put a mutation and a fluorescent marker into a few cell in mouse oesophageal epithelium in vivo. Over a year the mutant cells’ progeny took over almost the entire epithelium
Tumours are clones. The evidence is that all cells have the same gene changes, except for the most recent ones, Precursor clones, having some of the mutations seen in a tumour, can sometimes be detected in flanking tissue that is superficially normal
a) genetic instability in cancer cells
b) evidence that most cancers have specific defects that make them genetically unstable
a) most cancers appear to be genetically unstable (more prone than normal cells to undergo mutations). Cancers seem to arise when many genes are mutated, and acquiring these mutations is accelerated in cells that are genetically instable (have a defect in maintaining the fidelity of their DNA). This defect itself id a mutation. One of the mutations picked up by a cancer cell, probably early in its development, damages the DNA repair and maintenance machinery, so that subsequent mutations occur at a higher frequency
b) Best evidence is that individual cases of cancer show different kinds of genetic instability. This was first shown in colon cancers where there are at least two obvioiusly different types of instability. About 15% of tumours have normal or almost normal chromosomes, but show sequence instability (aka microsatellite instability), causes by mutations that inactivate DNA mismatch repair. The most cases have a lot of rearranged chromosomes, known as chromosomal instability (CIN), while generally having a near-normal rate of point mutation. These have mutations in the genes managing or repairing chromosomes. Some tumours have both kinds of instability. There are also other kinds of genetic instability
Mutations that cause genetic instability
a) overview of genes involved in causing genetic instability
b) DNA repair and mutations in DNA repair that give rise to genetic instability - main types of DNA damage and repair
a) Instability arises from defects in DNA repair or defects in replication and mitosis. For most cancers we don’t yet know what mutations give rise to genetic instability, but we do know the genes involved in a few specific cases. (see image)
b) DNA repair enzymes fix the chemical change, DNA strand breaks and crosslinks, and errors made by the DNA replication enzymes. Main categories are i) those that deal with damaged bases, including chemically modified bases and UV-induced pyrimidine dimers and either excise the base or the whole nucleotide. This isn’t known to be defective in human cancer except in people with inherited deficiency. ii) mismatch repair iii) two repair pathways, non homologous end joining (NHEJ) and homologous recombination (HR), that deal with DNA strand breaks and crosslinks between the strands
Mismatch repair, and instability
Deals with mismatched bases and also small loops that occur where polymerases slip while replicating repeats such as AAAAA, GTGTGT, or GTCGTCGTC, and add or delete a copy of the repeat, generating a tiny mismatched loop. If mismatch repair is defective, these slippage loops persist, and a striking effect is shrinkage or expansion of short repeats known as microsatellites (used for forensic DNA fingerprinting). Hence ‘microsatellite instability’ is a symptom of mismatch repair deficiency. There is also an associated higher rate of point-mutation because mismatched bases are not detected.
Failure of mismatch repair/microsatellite instability was the first example of genetic instability discovered. It occurs in about 15% sporadic colon cancers, usually by inactivation of MLH1 or MSH2, key components of mismatch repair machinery. These colon cancers have a roughly hundred-fold increased rate of small mutations compared to normal cells, including both single base changes (mismatches) and frameshifts caused by elongation or shortening of repeats. Both TGFβR II and Bax suffer such mutations. Incidentally, inactivation of MLH1 is quite often by epigenetic change, methylation of the DNA of its promoter, silencing its expression. Mismatch repair is also mutated in hereditary Lynch Syndrome
Repair of DNA strand breaks
Some chromosome instability is due to defects in DNA strand break repair. Several pathways tackle these important lesions: single strand break repair; double strand break repair by non-homologous end joining; and double strand break repair by homologous recombination.
Repair of double strand breaks by homologous recombination relies on there being two copies of the genome. It uses the other copy of the broken DNA sequence - usually the sister chromatid - as a template to re-synthesise the broken bit. Among the many proteins involved are the BRCA1 and BRCA2 genes/proteins, known for their role in hereditary breast cancer. (As with other heredetary pre-dispositions, in hereditary breast cancer patients, they inherit one damaged BRCA2 gene and the remaining copy is subsequently mutated in the developing cancer cell). The homologous recombination system, including BRCA1 and BRCA2, is also involved in repairing chemical crosslinking between DNA strands.
Mutations in other components of HR repair pathway can lead to genetic instability
Errors in DNA replication or mitosis
a) Mutations directly affecting DNA synthesis
b) Mutations causing defects in mitosis
c) mutations in cell cycle checkpoints
a) Mutations in DNA polymerase ε proof-reading domain can cause an extremely high error rate. Such mutations are found in a small percent of colorectal tumours
b) Another probable source of chromosome instability is errors in chromosome segregation. In some cancer cells, chromosomes sometimes get left behind or broken during anaphase. Lagging chromosomes can be seen in mitosis. The isolated chromosome may be lost during this cell division or inappropriately inherited leading to aneuploidy.
c) If DNA is damaged or replication encounters a problem, the cell cycle should be halted at a checkpoint, so defects in checkpoints may leave problems unresolved. So, for example, mutation of p53 protein may contribute to instability.
The Vogelstein model of colorectal cancer
Relates mutated genes to the stages of cancer development. It’s oversimplified, but provodes a helpful model of what might be happening
The first known change is mutation in either the APC or β-catenin gene, and may be required for adenoma formation. Mutating either gene has much the same effect, so these are alternatives. The suggested second mutation is genetic instability (ie mutation in CDC4 or CIN). Third is mutation in KRAS or BRAF. Fourth mutation is in PIK3CA or PTEN. Fifth is mutation in p53/TP53 or BAX. The last suggested mutation is in SMAD4 or TGF-βRII
Overview of hereditary predisposition to cancer
The affected individual has a high risk (usually 50% or higher) of developing cancer. The individual inherits one of the mutations required to get cancer. The inherited mutation is usually, but not always, in one copy of a tumour suppressor gene, rather than an ongogene. In ‘sporadic’ cases, two independent mutations in the same cell, would be required to inactivate this tumour suppressor gene. In the predisposed person, all their cells start with one copy inactivated and one intact copy, so cella behave normally. The second mutation is aquired in a cell during a person’s life which alters the cell’s behaviour
Why are hereditary predispositions to cancer important
They are among the commonest genetic diseases and they provided an important route to the discovery of tumour suppressor genes, including APC and RB1. At least a few percent of cancer cases is due to these predispositions, and the individuals affected may have a very high probability of developing a particular cancer (BRCA2 mutation gives a lifetime risk of 40-80% of breast cancer. Hereditary predisposition mutations can either be in growth control genes (eg APC, RB1) or in genetic instability genes (MLH1, MSH2, BRCA2)
Hereditary colon cancer
a) adenomatous polyposis coli
b) hereditary non-polyposis colon cancer
a) Familial Adenomatous Polyposis - Is rare (affecting 1/1000) in which individuals develop 1000s of polys (adenomas) in colon in late adolescence. Mapping the pattern of inheritance identified the responsible gene as APC. This is a tumour suppressor, and affected individuals inherit one mutant copy and lose the other after birth. Around 80% of sporadic colorectal cancers also have this mutation, but both copies are damaged after birth.
b) Lynch Syndrome - More common, 1% colon cancer cases. Mutations occur in one copy of MLH1 or MSH2. Both encode components of DNA mismatch repair machinery. ~15% sporadic cases have the same mutations, both mutations occurring after birth. The discovery of these genes was the first clear evidence that genetic instability was important in cancer
a) Hereditary predisposition to breast cancer
b) Inherited predisposition to retinoblastoma
a) Around 5% cases of breast cancer are individuals with strong hereditary predisposition. Perhaps half of these have a mutant copy of BRCA1 or BRCA2. These are unrelated proteins but are both components of DNA strand break repair. Loss of both copies of these genes gives genetic instability. However, neither gene is mutated very often in non-hereditary breast cancer. BRCA2 mutation also confers susceptibility to other cancers, including ovarian and prostate
b) Retinoblastoma is a rare tumour arising in the immature retina, in children under a few years old. About 40% of cases are hereditary, and on average predisposed individuals develop 3 tumours. The gene mutated is RB1, known to control the G1-S checkpoint.
Demonstration that genetic instability may be separated from growth control
Genetic instablility mutations are distinct from growth control mutations. This is demonstrated in reversion of BRCA2 mutations. BRCA2 is a component of HR repair. When cancers with BRCA2 mutations are treated with DNA-crosslinking agents, the treatment selects for cells that can repair the lesions, so selects for revertants of the BRCA2 mutation. These revertants have lost genetic instability, but still go on to kill the patient
