HC6: Cancer and genome integrity

Genome instability and mutation

Many cell divisions are required in embryonic development and during human life. Daughter cells inherit identical DNA. Cell division requires accurate DNA duplication and takes approximately 5 hours. 6 x 10⁹base pairs are copied almost flawlessly → there is less than 1 error per division.

Mutation repair:

If not repaired, DNA lesions can lead to mutations. Mutations can arise during regular DNA replication → DNA replication errors. Base mis-incorporation can occur, for example, a T can be inserted opposite of a G. Originally, there are 60.000 errors per cell division. However, there are several processes to correct such mistakes:

• Proofreading by DNA polymerase + exonuclease
  • Exonuclease activity makes it possible for DNA polymerase to misincorporate a base, go back to the former situation and try it again
  • Germline mutations in POLE and POLD1 (the main polymerases that replicate DNA) predispose to colorectal adenomas and carcinomas
  • Reduces misincorporation by DNA polymerase from 10^-5to 10^-7→ 600 errors per cell division
• Mismatch repair
  • Removes the newly synthesized strand, so that polymerase can try it again
    • Occurs if exonuclease has not been able to remove the incorrect base
  • Reduces the error rate during DNA replication from 10^-7 to 10^-9 or 10^-10 → there is less than 1 error per cell division on undamaged DNA

Lynch syndrome:

Lynch syndrome is a hereditary colon cancer. In case of Lynch syndrome, there is a mismatch repair deficiency. Several mutations predispose to developing Lynch syndrome, such as mutations in:

• MLH1
• MSH2
• MSH6
• PMS2

Replication of damaged DNA

Threats to genome stability:

Humans are continuously exposed to sources which damage DNA. DNA damaging agents can form threats to genome stability:

• Lifestyle
• Environmental or industrial
• Medical application
• Food source

Sunbathing:

In case of sunbathing there is exposure to UV-light. This causes replication blocks → a base is linked to another base → DNA polymerase stops because it cannot recognize a base. The DNA polymerase stops replicating → causes cell death.

A number of polymerases can bypass DNA damages → translesion synthesis polymerases (TLS). Because TLS can replicate damaged DNA, cell death is prevented. Sometimes these polymerases make errors, leading to a next round of replication for mutation.

POLη is the translesion polymerase bypassing mutation due to UV-damage. This is one of the most precise translesion polymerases → has evolved a lot. If another translesion polymerase bypasses the DNA damage, the chance of errors is higher.

A germline homozygous mutation of POLη leads to xeroderma pigmentosum variant-patients. Because other translesion polymerases have to do the work, these patients have sun-damaged skin and can easily develop skin cancer.

Endogenous sources:

Human cells also produce DNA damage every day:

• Production of free radicals
• Chemical instability
  • 10.000 bases per cell per day are lost
  • Depurination
    • Especially purines are prone to hydrolyse
    • The base is lost → suddenly there is no information during replication
  • Deamination: cytosine is methylated at its 5m position → a thymine is created
    • This is an epigenetic marker → changes the availability of chromatin to transcription factors

DNA damage repair

DNA damage repair pathways:

The only solution to deal with this is to repair the DNA damage before it is replicated. There are several types of DNA damage repair pathways:

• Base excision repair (BER)
  • For base modifications
    • Deal with small changes
• Nucleotide Excision Repair (NER)
  • For UV damage or chemical damages
    • For instance induced by cigarette smoke
• Double strand break repair (NHEJ/HR)
  • Breaks in both DNA strands
    • For instance induced by ionizing DNA sources
• ICL repair
  • Link 2 DNA-strands together → replication cannot occur
  • Patients with a deficiency for ICL repair develop Fanconi anemia

Human syndromes with defective genome maintenance:

Individuals with defective or reduced capacity to repair DNA damage are predisposed to:

• Developmental abnormalities
• Neurological abnormalities
• Immune deficiency
• Sensitivity to DNA damaging agents
• Increased cancer risk
• Premature aging

All these patients have a hugely increased risk of developing cancer. All mutations are homozygous → both alleles in all cells are mutated.

Heterozygous mutations

MMR gene mutation:

In a heterozygous mutation, only 1 allele is mutated. An example of a heterozygous mutation is MMR gene mutation. MMR gene mutation carriers are predisposed to develop Lynch syndrome. There is a recessive mutation on cell level, but the penetrance is high → it is a dominant trait. Almost all of these patients develop colon cancer before they are 40-years-old. Cells divide so frequently that the second allele is mutated, which enhances the mutation of the cell → increases the probability of cancer.

BRCA1/2 gene mutation:

BRCA1/2 gene mutation is also a heterozygous mutation. BRCA1/2 carriers are predisposed to develop breast cancer. BRCA1/2 play a large role in homologous recombination. If this doesn’t function properly, it leads to lots of breaks and translocations.

Cancer genome sequencing

Cancer genome sequencing can show which genes play a part in the development of cancer. There are 3 different types of cancer genome sequencing:

• Whole genome
  • Structural variants
  • Point mutations
  • Copy number variation
  • Everything is sequenced
• Whole-exome
  • Point mutations
  • Copy number variation
  • All the exons of all the genes are sequenced
• Gene panel sequencing
  • Diagnostics
  • Point mutations
  • Genes of which is known that they are frequently mutated in a certain type cancer are sequenced → it becomes visible whether there are mutations
  • Creates a specialized therapy to treat the patient

Driver and passenger mutations:

During sequencing, 2 types of mutations are found:

• Passengers
  • No advantage/disadvantage → mutations in genes which do not contribute to the cancer process
  • Occur at random
• Drivers
  • Growth/survival advantage
  • Nonsilent
  • Hit functional regions of cancer genes

Prevalence of somatic mutations

The prevalence of mutations varies among cancer types:

• In colorectal cancer, there are about 750 mutations per tumor
  • Mismatch repair is lost
• The amount of lung cancer and melanoma mutations is similar to colorectal cancer
  • The main cause for this is smoking
• Childhood and liquid cancers have a lower amount of mutations → fewer mutations are necessary
  • The cause for this is mainly unknown.

Nature of mutations

The nature of mutations varies among cancer types. The mutational spectrum of lung cancer is different than of melanoma. The contribution of mutagenic processes to different kinds of tumors varies → smoking causes lung cancer, UV-exposure causes skin cancer.

The mutational prolife of a cancer genome exposes tumor etiology. Cancer takes about 35 years to develop. Different kinds of exposures lead to a final mutational profile.

Mutational signatures:

Mutational signatures can reveal history of cancer development and may aid in therapy choice. Bioinformatic analysis of mutational spectra from large numbers of cancers into separate mutational signatures show every base change. There are 64 different possibilities and >45 mutational signatures have been identified. For instance, it can be shown that deamination of 5-methylcytosine leads to thymine generating G-T mismatches in DNA.

It is discovered what causes cancer in individuals. Different tumors in different tissues are caused by different mutational signatures.

SBS4:

Signature SBS4 has been found predominantly in:

• Lung adenocarcinoma
• Lung squamous carcinoma
• Small cell lung carcinoma

There are predominantly C-A mutations. Signature SBS4 is associated with smoking. Its prolife is similar to the mutational pattern observed in experimental systems exposed to tobacco carcinogens. Signature SBS4 is likely due to direct DNA damage by tobacco smoke mutagens.

SBS7a:

Signature SBS7a has been found predominantly in:

• Skin cancers
• Cancers of the lip categorized as head and neck or oral squamous cancers

Based on its prevalence in UV-exposed areas and the similarity of the mutational pattern to that observed in experimental systems exposed to ultraviolet signature SBS7a is likely due to ultraviolet light exposure.