Genes and Cancer

Question 1

What environmental factors that causes damage to DNA

Answer 1

Chemical substances (carcinogens of smoking)
Radiation (therapeutic, nuclear, atomic bomb survivors)
Viruses
-all associated with increased incidence of cancer

Question 2

Cancer genetics

Answer 2

• Cancers are induced by mutations in cancer causing genes (therefore consider cancer as a genetic disorder)
• Acquired or somatic mutations in majority (cannot be inherited)
• In some cases the mutations may be inherited (familial cancers) - mendelian (familial breast/colorectal cancer) + susceptibility genes

Question 3

Genes which cause cancer

Answer 3

Abnormal growth of tissue giving those cells a proliferative advantage
-Oncogenes
-Tumour Suppressor Genes
-Genes involved in regulation of apoptosis (cell death)
–>cells may not be reapidly proliferating, but impairment of normal death pathways
-Epigenetic changes
-Abnormalities of DNA repair genes
-Non coding RNA – e.g. microRNA (effect regulation of gene expression)
(mutations/changes in other pathways that contribute to development or progression of cancer)
Normal tissue: Homesostasis
Roads to Neoplasia: Proliferation –Neoplasia–> Death

Question 4

Ongogenes

Answer 4

Dominant acting genes – transform cells
(requires a mutation of a single allelle in the oncogene only to transform cells to give growth/proliferative advantage)
Initially recognized in viral induced tumours
Normal state = proto–oncogene
-switched on when cells divide and then switched off (carefully regulated)
Activated by mutation to oncogene (able to transform a cell)
Proto-ongogene –Mutation–> Ongogene

Question 5

Function of Oncogene

Answer 5

Most involved in signal transduction

• Growth factors
• Growth factor receptor (more common) (e.g. point mutation, causing it to be constitutively active in absence on ligand binding)
• Intracellular signal transmission
• Nuclear transcription factors

Question 6

Hyperactive growth control pathway

Answer 6

Growth factor –> Receptor –> Scanning Enzymes –> Cell Nucleus –> Transcription factors –> DNA –> Excessive Cell Proliferation

Question 7

Transform normal cells to neoplastic cells

Answer 7

Ongogenes transform Normal cells –> Neoplastic cell
-may grow independently of growth factors
Increased cell proliferation
Block normal cell differentiation/maturation
(malignant cells have increased proliferation but also immature (lack normal differentiation of epithelial cells)
Initiation event
Tumour progression

Question 8

What are the 2 main features of Malignant cells

Answer 8

Increased Proliferation

Immature (lack normal differentiation of epithelial cells)

Question 9

Diagram of activation of Oncogenes

Answer 9

Proto-oncogene —> (environmental) Cancer causing (UV light, chemicals etc) –> Oncogene –> Cancerous Phenotype

Question 10

Activation of Oncogenes (molecular level changes)

Answer 10

1. Gene amplification
2. Over expression of gene (change promotor to make more mRNA and hence more Protein)
3. Point mutations
   (change critical base pairing, change a/acid, resulting in sig. change in function of proto-oncogene)
4. Chromosome Translocations
   (swapping of chromosome material, and if involving proto-oncogene, can lead to activation to an oncogene)

Question 11

Amplification of Onocogenes

Answer 11

1. Neuroblastoma (paediatric tumour, will respond to chemo)
2. N-myc amplification (nuclear transcriptional regulator of a number of critical genes)
3. Unfavourable prognosis (amplification fo N-myc is a prognositc factor that carriers worse prognosis and would alter treatment if present)
   - Increased gene copy number

Question 12

FISH of Amplification of Oncogenes

Answer 12

DNA probe that binds to the neuroblastoma proto-oncogene
labelled with red fluorescent probe
let hybridise onto cells
looked at under fluorescent microscope
Amplification of N-myc proto-oncogene = multiple (more than 2) dots
-gives pathogenesis of tumour
-gives clinical info that alters therapy

Question 13

HER-2 amplification in breast cancer

Answer 13

Red signal = HER-2 gene (on chromosome 17)
-look for its amplification on FISH study
Green signal = ch 17 centromere
Amplification = more than 2 genes

Question 14

Clinical significance of HER-2 amplification

Answer 14

HER-2 is a proto-oncogene - it encodes for an epidermal growth factor receptor in mammary cells (ETFr)
The HER-2 protein is over-expressed in 30% of breast cancer patients.
In 90% of cases, this is due to amplification of the HER-2 gene (pathogenesis. not initiating event but important in progression)
HER-2 is a target for the antibody herceptin (targeted antibody therapy)
-important part of therapy with cytotoxic drugs

Question 15

Where is the HER-2 gene located?

Answer 15

Chromosome 17

Question 16

Clinical significance of HER-2 amplification Diagram

Answer 16

HER-2 Gene amplification in Breast Cancer (chromosome 17 (normal))

• -> HER-2 gene amplification, 20-30% of breast cancer patients
• -> Increased expression of HER-2 mRNA
• -> Increased expression of HER-2 protein
• -> Signals cels to proliferate, tumours more aggressive –> Breast cancer cell

Question 17

HER-2 Targeted Therapy

Answer 17

HER2 oncogene amplification in the tumour cell changes RNA and results in HER2 protein over expression
Herceptin targets HER2 Epidermal growth factor Receptor-2 protein
-binds and blocks off so EGF cannot bind

Question 18

HER2 and EGF meaning

Answer 18

HER2= Human Epidermal growth factor Receptor-2
EGF= Epidermal Growth Factor

Question 19

Activation of oncogenes: Over expression of gene

Answer 19

Results in increased mRNA and hence increased protein expression

• normal copy number of genes
• but mutations/changes in promotor/regulatory regions will result in increased copy number

Question 20

Activation of oncogenes: Point mutations

Answer 20

Quite common
Single base change, but if is a change of a critical a/acid, can lead to constitutive activation of a signalling pathway
Mutations within EGF receptor allows it to be active without binding its ligand, activating down stream signalling pathways –> Gene transcription –> Cell cycle activation
–> Increased Survival, proliferation and invasion(into lymphatics and BV)

Question 21

How do chromosome translocations activate proto-oncogenes ?

Answer 21

(if chromosome translocation brings 2 genes together in 5 prime 3 prime configuration) 1. Form fusion gene (-->fusion mRNA) which encodes for novel protein with increased activity
eg t(9;22) in chronic myeloid leukaemia
2. More common. Increased expression of oncogene e.g. by translocation moving adjacent to actively transcribed gene or promoter region (constitutively active) e.g. the immunoglobulin gene in B cell cancers (would result in up-regulation/increased expression of the proto-oncogene)

Question 22

Myc translocations in Burkitts Lymphoma

Answer 22

Lymphoma= cancer of lymphoid tissue
Burkitts Lymphoma is one of the most aggressive cancers
-cells developing every 24-48hrs
presented with abdominal pain
-mass which is dark centrally (necrotic/outgrown blood supply)
-biopsy chows Burkitts Lymphoma
–Monomorphic population of immature cells
-dark, alot of mitotic figures, actively dividing
-90% of Burkitts Lymphoma will have Chromosome translocation/abnormaltion (most common is c 8 is attached to 14) (some 8 and 22. others 2 and 8)

Question 23

Significance of Myc translocation in Burkitts Lymphoma

Answer 23

Long arm of Chromosome 8 contains protooncogene myc
-seen in neuroblastoma
-myc codes for a transcriptional factor
Chromosome 14 contains IgHeavy chain gene
Chromosome 2 and 22 contain IgLight chain genes
Translocations: takes myc protooncogene and places into 5’ to 3’ configuration with Ig gene
-since in lymphoid cell, Ig gene promotor regions are always activation –> upregulation of expression of myc onocogene –> increased transcription of other downstream genes

Question 24

Tumour Suppressor Genes

Answer 24

Recessive acting or “anti-oncogenes”
-regulate cell growth (“suppressors”)
Requires loss of activity – of both alleles (mutation of both allelles for TSG to contribute to malignant phenotype)
Often mechanism in familial cancers – one inherited mutation in TS gene with second acquired mutation causing loss of activity of the other allele

Question 25

Tumour Suppressor genes Accelerators vs Brakes

Answer 25

Accelerators (oncogenes): Substances that encourage growth --> Substances that trigger cell division Brakes (TSG): Substances that prevent growth --> Substances that stop cell division

Question 26

Tumour suppressor Gene diagram

Answer 26

Wild type TSG --New Mutation --> First "hit" (e.g. a mutation in one copy of the TSG) --Deletion--> Second "hit" (e.g. a deletion in the other copy of the TSG) --> Progression to tumour formation Nots: Frequently implicated in familial cancers (First "hit" (the germline mutation) is an Inherited Germline mutation in the TEG -compatible with life, as doesnt result in malignant phenotype, until mutation effecting the other allele, --> progression to tumour

Question 27

Retinoblastoma

Answer 27

“Two hit hypothesis” Paediatric tumour Rare 2x types: Hereditary or Non-Hereditary 1. Hereditary: Family history, Autosomal Dominant. Bilateral (both eyes, or two tumours in one eye). Early onset. Germline-mutation of Retinoblastoma gene.2nd hit resulted in tumours (all cels predisposed so not unexpected to multifocal tumours within one cell) 2. Non Hereditary: Sporadic form. Unilateral. Late Onset. later childhood). Retinoblastoma gene was normal/wild type. required 2x somatic/acquired hits within the same cell, before tumour developed. (single tumour and late onset) Retinoblastoma gene on Chromosome 13 is a cell cycle regulator, inherited form had a germline mutation. (every cell had this mutation)

Question 28

Apoptosis - Programmed Cell death

Answer 28

a) Normal Tissue in --> Homeostasis --> Out b) The roads to Neoplasia -Increased proliferation --> Neoplasia --> Death -Proliferation --> Neoplasia --> Death (arent giving cell proliferative advantage, the cell just dont die by normal programmed cell death pathway)

Question 29

Genes which affect apoptosis

Answer 29

1. Upregulation of genes which encode for proteins that block apoptosis - make "blocking" cell death pathway proteins. Single mutation 2. Loss of activity of genes which encode for proteins that mediate apoptosis eg fas, p53 - recessive action - have to lose both alleles

Question 30

Pathways of Apoptosis

Answer 30

1. Stress pathway Cell damage, activation of oncogenes, growth factor deprivation 2. Death receptor Pathway: Ligands (Fast, TRAIL, TNF) - ----> Cell Death

Question 31

Summary: Genes in cancer

Answer 31

1. Oncogenes - Dominant acting - Promote cell growth 2. Tumour suppressor genes - Loss of activity - Inhibit cell proliferation 3. Genes involved in apoptosis

Question 32

Multistep process

Answer 32

Cancer is a multistep process... Most cancers involve multiple genetic events or hits before the full cancer phenotype develops Mutation inactivates tumour suppressor gene --> Cells Proliferate --> Mutation inactivates DNA repair gene --> Mutation of proto-oncogene creates oncogene --> Mutation inactivates several more tumour supressor genes --> Cancer

Question 33

Epigenetic changes in cancer

Answer 33

Not mutation of cancer causing genes. Rather the way DNA is packaged. Effects Methylation and status. + the role of Non-coding RNA related to packaging

Question 34

What is the clinical relevance ? (Personalized medicine based on cancer genomics)

Answer 34

Personalized medicine based on cancer genomics • Molecular diagnosis and classification of tumours (molecular markers) (HER2 +ve cancer) (B2 melanoma) • Prognostic markers (Nmch amplification) • Targets for therapy • Markers to monitor response to therapy • Biomarkers eg cell free DNA

Question 35

What is the clinical relevance ? (Application to familial cancer)

Answer 35

Application to familial cancer Understanding molecular genetics allows introduction of predictive / at risk genetic testing Allows screening strategies, potential to reduce other risk factors, prophylactic surgery

Question 36

What is the clinical relevance Cycle

Answer 36

Genome technologies --> "Individualised" profile of cancer alterations --> Target therapy trials (Cancer genome alterations) --> Diagnosis and prognosis for individual patients -->

Question 37

Cancer Therapies that target oncogenic proteins

Answer 37

``` Anticancer Drug --> Disease Monocolonal antibodies: Trastuzumab (Herceptin) --> Breast Cancer Small Molecules: Imatinib --> Leukemia markers ```

Question 38

Chronic Myeloid Leukaemia (CML)

Answer 38

Clinical features: • High white blood cell count • Splenomegaly • Chromosomal abnormality -all have The Philadelphia Chromosome: t(9;22) Translocation -Protoncogene 9's c abl (weak tyrosine kinase) into a 5' to 3' configuration with 22's bcr gene --> bcr-abl --> Fusion protein with very potent tyrosine kinase activity

Question 39

The Philadelphia Chromosome:

Answer 39

Tyrosine kinases are present in most cancers and involved in signalling pathways Acquired chromosomal translocation seen in chronic myeloid leukaemia Clinical Implications: -Diagnostic criteria for CML (required) -Leukaemogeneic (will develop leuakemia) -Target for therapy -Biomarker to monitor response to therapy

Question 40

Bcr-Abl as a Therapeutic Target

Answer 40

Bcr-abl uses ATP to phosphorylate tyrosine residues switches on down stream signalling pathways making the cells proliferate Imatinib binds into ATP binding pocket, stopping proliferation, cells die via apoptosis

Question 41

Imatinib (glivec) 5 year data

Answer 41

Clinical remission 98% Cytogenetic remission 87% Overall survival at 5 yrs 89% (95%) Pre – Imatinib median survival CML 5 years Patients in complete cytogenetic remission > 2 years -chemo therapy half dead in 4 years

Question 42

Chronic Myeloid Leukaemia

Answer 42

Model for genetics and therapy of cancer - Acquired cytogenetic / molecular abnormality - Results in proto-oncogene activation with formation of a potent tyrosine kinase - Key pathogenic event present in leukaemic but not normal cells - First example of targeted cancer therapy