HC2: Cancer genetics

Multistage evolution of cancer

A normal epithelial cell has to go through several stages to be transformed into a metastasizing tumor. There is a progressive development from normal to neoplastic tissue, with many non-malignant intermediate stages.

Clonal expansion model:

Cancer development is driven by the accumulation of mutations:

1. An initiating mutation occurs in a normal cell → gives the cell a growth advantage over his neighboring cells
2. During life, a second mutation occurs → accelerates growth further
3. A third and fourth mutation occurs → formation of a tumor

Types of mutations and their biological consequences

A mutation is a permanent alteration in a parental DNA-sequence → a cell or organism. If it is in a parental organism, it is a hereditary mutation → is transferred to a child. If it occurs in a cell, the mutation is transmitted to the daughter cells.

Classes of mutations:

There are two classes of mutations:

• Chromosome mutations: may affect the expression of many genes
  • Chromosome losses/gains
  • Translocations
  • Multi-locus deletions
• Gene mutations: may affect the expression of a few genes
  • Deletions/insertions
  • Base pair substitutions
  • Frameshift mutations

Chromosome mutations

Multi-locus deletions:

In case of multi-locus deletions, a fairly big part of a chromosome is deleted. This leads to a loss of function of the deleted alleles. Deletion on an autosomal chromosome leads to hemizygosity for multiple genes → only copy of the allele remains. Hemizygosity means that only 1 copy of the gene is left in 1 cell.

Intragenic mutations:

If there is a gene that consists of 5 exons, and there is a deletion of exon 2 and another deletion of exon 2 and 3, the mutation is intragenic. This doesn’t necessarily lead to loss of function, but most of the time, it does.

This has mutagenic consequences:

• If exon 2 is deleted, the reading frame doesn’t change → even though the protein may be smaller because it lacks a certain part, the total-protein will remain functional or at least partially functional
  • Exon 2 isn’t necessary for the function of the total protein
• In case of exon 2+3 deletion, the reading frame does change → there is introduction of a stop codon → the protein becomes truncated and loses its function

Gene mutations

Base pair mutations over a gene can be distinguished in mutations which occur in:

• Intron: most often have no consequences
• Promoter: may affect transcription efficiency
  • Either increase or diminish
• Splice site: may affect splicing
  • E.g. can cause exon skipping
• Exon: may affect protein composition

In a splicing consensus of intronic and exonic sequences the following is visible:

• Intronic sequences always start with GT (GU in mRNA)
• Intronic sequences always end with AG

If this GT sequence is changed into an AT sequence, the exon isn’t recognized anymore → cannot encode its information into a protein. The same thing happens at the other end of the intron.

Base pair substitutions:

A certain wild type gene starts with GGG GAA GTA → encodes for glycine, glutaminic acid and valine. A frameshift mutation leads to introduction of a stop codon very early in the gene → loss of function.

If the GC base pair is changed into a TA base pair, it becomes TA → a TAA sequence is introduced, and TAA is a stop codon. This also leads to protein truncation and loss of function. This is a nonsense mutation.

If the base pair mutation is from GC to CG, another amino acid than glutaminic acid will be synthesized. This is a missense mutation, a non-synonymous amino acid change because glutaminic acid (an acidic amino acid) is exchanged for glutamine (a basic amino acid). It is hard to predict whether this will lead to a loss of function.

Functional impact:

If a mutation occurs in evolutionary conserved regions of the gene, there is lots of selective pressure because especially these domains have to be maintained → are important for protein-function. If a mutation occurs, there is a chance that it’ll lead to:

• Change of function
• Loss of function
• Partial function

Gene mutations in cancer

A normal epithelial cell needs approximately 7 mutations to change into a malignant tumor → 7 mutations are needed to convert a normal epithelial cell into an invasive carcinoma.

Mutation rate:

The mutation rate for a gene in somatic cells is approximately 1:10^-7/cell division:

• There are 10^13-14 cells in the human body
• The chance for specific independent mutations in a cell is 1:10^-42
• 10¹⁶ cells turn over during human life

These numbers indicate that the chance of getting cancer is nihil → the mutation rate is very low. Cancer still exists, because:

• Some mutations cause genomic instability → increase the mutation rate
• The mutation rate for some types of mutation is much higher than 1:10^-7
• Other mechanisms than mutation can impair gene function → epigenetic silencing of genes
• Some people carry predisposing mutations

Cancer genes:

During cancer development, 2 classes of genes can be mutated:

• Oncogenes
• Tumor suppressing genes

Oncogenes

Some cancer viruses contain a copy of a normal cellular gene → proto-oncogenes. Some cancer viruses contain an activated form → oncogenes. A gene is only called an oncogene when it has become mutated and starts contributing to cancer development. An oncogene is an activated form. There is a highly specific mutation in only 1 allele → the phenotype is dominant.

Oncogene activation:

Activation of oncogenes results in “gain of function”:

1. A proto-oncogene is present
2. An active mutation occurs → functional product
   • Coding mutation: results in an abnormal protein
   • Mutation in regulatory sequences: results in an excessive amount of protein
   • Translocation of genes: results in a novel hybrid protein → an activated form
   • Hybrid protein: a product of 2 different genes
   • Genetic amplification: results in a protein being produced in massive amounts
   • This occurs quite frequently

In conclusion, there are 2 ways to get oncogenic activation:

• Production of a protein which is abnormally active
• Production of much more of the normal protein

Oncogenic Ras activation:

The Ras oncogene is an oncogene which is frequently mutated in cancer. Normally, in inactive state, Ras is bound to GDP. It can be activated as follows:

1. There are upstream stimulatory signals which signal that the cell has to start dividing
   • The happens because Ras is a gene which is involved in growth control
2. GEF exchanges GDP forGTP → Ras is activated
   • GEF: guanine nucleotide exchange factor
3. Activated Ras starts downstream signaling → tells kinases to kinase their targets → stimulate cell growth
4. Normally, if the growth factors disappear, GAP binds to Ras
   • GAP: GTPase activating protein
5. GAP binding causes a 1000x increase in GTPase activity → Ras inactivation
   • The GTPase activity is an intrinsic characteristic of Ras

If there is a mutation in Ras which blocks the binding of GAP, Ras stays “on” and cannot be switched “off”. In this case Ras continuously stimulates cell growth → tumor growth.

Gene amplification:

Oncogenes can be activated by gene amplification. Amplification of a normal gene occurs >100x:

• Double minutes: pieces of DNA which are all identical and which contain an oncogene
• Homogeneously staining regions (HSRs): the gene lands on another chromosome and inhabits almost half of it

Examples of oncogenes which become activated because of gene amplification are:

• N-Myc gene: frequently amplified in neuroblastomas
• ERBB2 genes: frequently amplified in breast, ovarian, gastric, colon and lung cancer

Chromosomal translocation:

A chromosomal translocation where a normal protein is overproduced occurs in 85% of all Burkitt lymphoma cases:

1. There is a translocation between chromosome 8 and 14
2. After the translocation IgH and MYC are translocated next to each other → MYC is not controlled by its own promotor anymore, but by the IgH promotor instead
3. In B-cells which produce lots of IgH, lots of MYC is produced
   • MYC is a growth factor and an oncogenic product
   • There is an up-regulated expression of the structurally normal MYC protein
   • Exon 1 is noncoding

Chromosomal translocation where genes are fused occurs in >95% of all chronic leukemia patients:

1. There is a translocation between chromosome 9 and 22
2. A hybrid chimeric fusion gene which is constantly active is made
   • This is not an oncogene which is prescribed by the promotor of another gene
3. The gene is constantly signaling → cannot be switched off anymore → constitutionally active tyrosine kinase

Tumor suppressor genes

Tumor suppressor activity in normal cells:

If human tumor cells are injected in immune deficient mice, the cells will cause tumor formations in the mice. If normal human cells are given to immune deficient mice, there is no tumor formation → normal human cells may have the capacity to suppress the tumorigenic potency of tumor cells.

A tetraploid cell hybrid can be made by fusing human tumor cells and human normal cells. If this cell hybrid is injected in immune deficient mice, there is no tumor formation. Normal human cells apparently have the capacity to suppress tumorigenic capacity of tumor cells. These genes are called tumor suppressor genes.

2 hit-hypothesis of Knudsen:

Retinoblastoma (RB) is a type of eye cancer which mainly develops in children. Children who have the tumor in both eyes develop tumors much faster than children who have the tumor in only 1 eye. A possible explanation for this is the 2 hit-hypothesis of Knudsen:

• Sporadic, unilateral cases: a first and second hit of a mutation is necessary in order to develop a tumor
• Familial, bilateral cases: 1 of the parents already has the mutation in the RB gene → the mutation is inherited by the child → the child has only 1 functional copy of RB in all cells
  • The chance that the second allele is lost is very high → leas to development of an eye tumor

Loss of tumor suppressor gene activity:

If there is 1 chromosome where RB is mutated, and 1 chromosome with functional RB, this doesn’t result in oncogenic dysfunction → the mutation cannot take place in an oncogene, because 1 mutated oncogene already leads to dysfunction. However, loss of the second allele does lead to dysfunction → it is a tumor suppressor gene. In case of tumor suppressor genes, expression of the second allele also needs to be lost.

Epigenetic silencing

In case of epigenetic silencing, cytosines become methylated at the 5’ position. Especially the CpG-sites are prone to become methylated by methylases. If the promotor regions of genes are methylated, the DNA gets a more closed confirmation → inhibition of transcription → gene is silenced:

• Hypermethylation: if tumor suppressor genes are methylated, this will lead to loss of expression
• Hypomethylation: if oncogenes in normal cells lose methylation it becomes activated → the gene is expressed in a situation where it should not be expressed

Mutational spectra of cancer genes

Mutations are recessive in nature. Many hereditary forms of cancer start off with a situation where all body cells have already lost expression of one allele.

Mutational spectra of oncogenes and tumor suppressor genes differ:

• Oncogenes: mutations are dominant, causing gain of function
  • Missense mutations
    • Are predominantly visible
    • Change the amino-acid composition
  • Mutational hotspots
    • May for example lead to GTPase inactivation of Ras
• Tumor suppressor genes: mutations are recessive, causing loss of function
  • Truncating mutations
    • Over the whole gene
    • Almost any truncation of the protein will lead to loss of function

Recurrently mutated cancer genes:

Mutations can be frequent and common, but also rare and highly specific. There are more than 200 recurrently mutated cancer genes → drivers. These genes take part in many kinds of signaling pathways:

• TP53 gene: mutated in almost any kind of tumor
  • Except for AML and kidney cancer
  • A very common gene to be mutated
• VHL gene: is only mutated in 50% of kidney cancer cases

Because there is difference between the genes that need to be mutated in order to be transformed from a normal cell into a tumor cell, cancer treatment is very difficult.

Co-occurrence of oncogenic mutations:

Co-occurrence of oncogenic mutations can occur → there may be a mutation in gene A and a mutation in gene B. This can by caused by:

• The genes having synergestic effects → the effect together is stronger than the effect separate
• The genes ameliorate deleterious effects → overcome deleterious effects of the other gene
• The effect of mutational processes

Mutations may also be mutually exclusive → tumors with mutations in gene A hardly have mutations in gene B. This can be caused by:

• The genes being functionally redundant → if a function has already been gained by gene A, it doesn’t have to be gained by gene B
• The genes being synthetically lethal if they come together → if 2 mutated genes come together, the cell dies
• The genes occurring in different cancer subtypes

If there isn’t any bias against or in favor of 2 genes being mutated, there’s a random assortment and independence of effects.

KRAS mutations rarely occur together with BRAF mutations, but frequently occur with mutations in STK11. The same concept can be applied for TP53.