Intro + Oncogenes

Question 1

How is cell division controlled?

Answer 1

= TIGHTLY!

Cells normally only divide in response to extracellular signals (growth factors)

Unwanted cells lost through apoptosis, anoikis

Processes (e.g. cell growth / anti-growth, cell death / survival)
= controlled by :
Tumour suppressor genes
Proto-oncogenes

Question 2

What does loss of control over cell division cause?

Answer 2

Tumour Growth

Mutations that cause loss of tumour suppressor genes / gain of function of protooncogenes
= tip balance towards HYPERPROLIFERATION

Question 3

What are oncogenes?

Answer 3

A gene whose presence results in a cancerous phenotype

Dominant at cellular genetic level (only need 1 copy)

Derived from genes normally involved in regulating proliferation and survival (proto-oncogenes) through either mutation or misregulation

Question 4

How were cellular oncogenes identified?

Answer 4

Originally thought to be infectious / viral disease

Transfection Protocol.

1. Chemically transformed mouse fibroblasts
2. DNA transfection using calcium phosphate co-preciptation procedure (crystals forms)
3. Add to Normal mouse fibroblasts (crystals taken up)
4. Formation of a focus (cancerous phenotype) of morphologically transformed cells
5. Injection of morphologically transformed cells into mouse host
6. Xenograph - forms tumour on mouse

= proved genetic origin

Question 5

What are some of the mechanisms for oncogene activation (from proto-oncogene)?

Answer 5

Deletion / Point mutation in coding sequence
= hyperactive protein made in normal amounts

Regulatory mutation
= normal protein greatly overproduced

Gene Amplification
= normal protein greatly overproduced

Chromosome Rearrangement
= nearby regulatory DNA sequences causes normal protein to be overproduced
OR
= fusion to actively transcribed gene produces hyperactive fusion protein

(Extra Reading)
= can also have Viral integration
- some viruses integrate DNA into host genome and activate proto-oncogenes (e.g. HPV, EBV)

= epigenetic alterations
- e.g. DNA methylation and histone modification
- can lead to activation of proto-oncogenes by altering their expression

Question 6

What the (7) type of oncogenes?

Answer 6

Can derive from following growth regualtory genes (proto-oncogenes):

1. Growth factors
2. Growth factor receptors (e.g. Receptor tyrosine kinases)
3. G proteins
4. Intracellular serine/ threonine kinases
5. Intracellular tyrosine kinases
6. Transcription factors
7. Negative regulators of apoptosis (e.g. Bcl)

Question 7

Autocrine signalling in cancer? (Growth factors as oncogenes) + examples?

Answer 7

Normal cells = paracrine or endocrine signalling
= cells do NOT produced own growth factors

Cancer cells = autocrine signalling
= produce own growth factor through mutation
= uncontrolled growth

e.g. TGFα
= ligand of epidermal growth factor receptor (EGFR)
= produced by lung, prostate, pancreatic, mesothelioma and breast cancer

Other e.g. (+receptor)
= SCF (Kit)
= VEGF-A (VEGF-R)
= HGF (Met)
= NRG (HER2/HER3)

Question 8

How does deregulation of receptor firing happen in cancer? (growth factor receptor as oncogenes)

Answer 8

Normal cell
= ligand-dependent firing
= dimerisation = activates kinase activity

Cancer cell
= ligand-independent firing
= through mutations affecting structure or overexpression
= can dimerise without ligand , activating kinase activity

overexpression e.g. = HER2 oncogene and breast cancer

Question 9

What are G-proteins and example? (G proteins as oncogenes)

Answer 9

= large family of proteins activated by binding GTP

= subfamily - monomerical small GTPases e.g. Ras

= bind GTP = on
= bind GDP = off (in cancer can not turn off)

= activated by GEFs (guanaine nucleotide exchange factors)

= inactivated by GAPs (GTPase activating proteins)

= Ras proteins: HRAS, NRAS, KRAS4A, KRAS4B = encoded by 3 genes

(Extra Reading)
= other examples
- Gαq = cell growth and division = breast, liver, colon
- Gαs = cAMP production and PKA activation = thyroid, adrenal cortical carcinoma
-Gα12/13 = regulate cell shape and motility = gastric cancer, hepatocellular carcinoma

Question 10

What do adaptor proteins do? (G proteins as oncogenes)

Answer 10

link receptor tyrosine kinase firing to RAS

Question 11

Examples of Ras mutations in cancer?

Answer 11

KRAS most common
= pancreatic (57%), colorectal (33%), biliary tract (31%), small intestine (20%), lung (17%), ovary (14%), endometrium (14%)
= usually codons 12, 13, 61

NRAS
= skin (18%), haemopoietic (10%), thyroid (8%)
= usually codons 12,13, 61

HRAS
= cervical (9%), head and neck (15%)
= usually codon 12

= activate downstream signalling pathways involved in cell growth and survival

Question 12

Ras Mutation Hotspots?

Answer 12

= codon 12 most common site of point mutation

= most mutations are gain of function

= mutational hotspots correlate with decreased GTPase activity

= conformational change

Question 13

G proteins and Intracellular serine / threonine kinases as oncogenes?

Answer 13

Mutant RAS / downstream serine / threonine kinases
= lead to abnormal proliferation

Mutations in downstream components of cascade also occur
= B-raf V600E and V600K mutations seen in melanoma

Question 14

Intracellular tyrosine kinases as oncogenes? The bcr-abl oncogene?

Answer 14

Translocation between chromosomes 9 and 22
= the Philadelphia chromosome
= and the bcr-abl oncogene

= encodes Bcr-abl fusion protein (= constitutively active tyrosine kinase)

= found in >95% of chronic myelogenous leukaemia (CML)

Question 15

How does bcr-abl oncogene cause cancer?

Answer 15

There is dimerisation

= transactivation

= becomes permanently switched on

= causes defects in many signalling pathways which drive phenotype of CML (+ small subset of ALL)

= results in proliferation and survival, transcription, proliferation, survival
(hallmarks of cancer)

= e.g. of downstream signalling pathways activated by BCR-ABL
= RAS-MAPK, PI3K-AKT

= also disrupts normal DNA repair mechanisms and increased genomic instability of leukemic cells

Question 16

Transcription factors as oncogenes? Myc transcription factor?

Answer 16

Myc
= regulates expression of genes involved in promoting cell proliferation and survival
= belongs to family of basic helix-loop-helix leucine zipper transcription factors that bind to DNA and regulate expression of target genes

Becomes oncogene in 2 ways:
1. myc locus amplified frequently in various leukaemia and carcinomas (10-50%)
2. expression of myc deregulated in Burkitt’s lymphoma by chromosomal translocation

> 70% cancers overexpress Myc or one of its homologues (N-Myc, L-Myc)

(Extra Reading)
= Myc promotes uptake of glucose and production of ATP through aerobic glycolysis (Warburg effect)
= promotes cell survival sometime BUT induced apoptosis in others
= regulates differentiation of many cell types
= stimulate cell proliferation by promoting G1 to S phase transition of cell cycle