PCR Application: Can Breast Cancer be Cured?

Normal, Healthy Cells Cells can change or differentiate to become specialised according to the tissue that they make up A healthy cell performs the correct functions according to its type and specialised state This feature of cells is controlled by our DNA

Cell division Cell specialisation/differentiation Cell adhesion Normal, Healthy Cells

Very common to get DNA mutations How does a healthy cell deal with this? Repair DNA e.g. BRCA2 protein Cell suicide – removes cells with badly damaged DNA When things go wrong

Normal, Healthy Cells

DNA Mutations Mutations are a change in the base nucleotide sequence of our DNA e.g. Deletion A T G C G A T  A T C G T T e.g. Insertion A T G C G A  A T G G C G A G Changes in the DNA sequence can change the amino acid sequence of the protein.

DNA Mutations If a gene becomes mutated, cells can start to lose control and become cancerous This can lead to the formation of a tumour

Cancer Cells Uncontrollable cell division Reduced cell death - cancer cells do not always respond to signals telling them to die No cell specialisation - cancer cells lose their specialised features Reduced cell adhesion - cancer cells lose their ability to stick together Cancer cells arise from genetic mutations

Cancerous Cells

External Factors causing genetic mutations Physical Carcinogens E.g. UV Radiation from sunlight Biological Carcinogens E.g. viruses such as HPV can create an opportunistic environment for cancer Chemical Carcinogens E.g. Tobacco, alcohol

Inheritance of these genetic mutations External Factors causing genetic mutations

Genotype Genetic makeup of a cell – the set of alleles that a person has. Phenotype The observable (way it looks) physical or biochemical appearance of an organism. Determined by genotype and environmental influences.

TYPICAL FORM MUTANT FORM (“WILD TYPE” - WT) Example

Genotype Terminology Heterozygous – when the cells contains 2 different alleles at a gene locus e.g. Bb Homozygous – when identical alleles of the gene are present on both homologous chromosomes e.g. BB or bb

Inherited Mutations The BRCA2 gene codes for a protein that repairs DNA. Some people can inherit a BRCA2 mutation (Bb genotype for BRCA2) These people are more susceptible to accumulating DNA damage and are consequently at a higher risk of breast cancer Tumours arise from cells where Bb has spontaneously mutated to become bb with time (since this means that they have no BRCA2 in these cells)

What Does Higher Risk of Breast Cancer Mean? 12% of women without any inherited mutations will develop breast cancer in their lives 45% of women with a harmful BRCA2 mutation will develop breast cancer by 70 years of age

Managing Risk Angelina Jolie has an inherited BRCA2 mutation and elected to have her breasts removed as a preventative measure

How did she know? A DNA sample would have been taken (from her blood or saliva) and this sample would have been sent to a testing laboratory Then the BRCA2 gene locus would have been amplified by PCR and her genotype analysed by gel electrophoresis

Managing Risk Other options; Enhanced screening – frequent mammograms Chemoprevention – possibly tamoxifen or ralaxifene may help to reduce risk, but this has not yet been proven

How can we kill cancer cells with BRCA2 Mutations? Cancer cell’s repair mechanisms cannot cope Cancer cells commit suicide (Healthy cells also commit suicide)

The Problem with mutated BRCA The mutation in the BRCA2 (bb) gene in tumour cells is not enough to induce cell suicide So BRCA2 mutated cells do not respond very well to chemotherapy or radiotherapy This is because other DNA repair mechanisms exist in the body. The other major one is the PARP pathway

Overcoming this Problem Treating cancers caused by BRCA2 mutations by inhibiting the other DNA repair pathway: Synthetic Lethality BRCA PARP BRCA PARP BRCA PARP BRCA PARP Cell survives Cell survives Cell survives Cell Death BRCA2 Mutant Cell Normal Cell Normal Cell with PARP inhibitor drug BRCA2 mutant cell with PARP inhibitor drug DNA Damage – chemotherapy/radiotherapy

Advantage of synthetic lethality– the person’s healthy cells are not damaged – only their cancer cells BRCA PARP BRCA PARP Cell survives Cell Death Normal Cell with PARP inhibitor drug BRCA2 mutant cell with PARP inhibitor drug DNA Damage Conventional chemotherapy and radiotherapy both cause a lot of damage to healthy cells

Synthetic Lethality NO BRCA2 + NO other = CELL DEATH mechanisms of cancer cells of DNA repair + Chemotherapy or Radiotherapy Synthetic lethal treatments are currently being researched in Cardiff University and elsewhere as a potential new therapy for breast cancer INHERITED MUTATION + PARP INHIBITOR DRUG

Your Task Your PCR samples are from 4 individuals with or without BRCA2 mutations Your tasks are to: 1.GENOTYPE each sample 2.PHENOTYPE each sample