Oncogenes Flashcards
In which ways were oncogenes discovered?
1) studies of retroviruses/homology to viral proteins
2) 3T3 transformation assay
3) chromosomal amplifications and rearrangements
4) DNA sequencing
Outline the discovery of oncogenes in RSV
1) sarcoma removed from chicken
2) sarcoma ground up and added to sand
3) collect filtrate that has passed through fine pore filter
4) inject filtrate into young chicken
5) observe sarcoma in injected chicken
How does avian leukosis virus differ from rous sarcoma virus?
RSV has src oncogene
What is src?
Protein tyrosine kinase that phosphorylase targets
Mutant form of v-src that is truncated to remove SH3 domain. This prevents the domain from folding up and leaves the kinase domain exposed
What are the functions of src?
Binds to and activates signalling pathways
Involved in growth induced mitogenic signalling, survival and migration
What is meant by the oncogene hypothesis?
A normal cellular gene (proto-oncogene) can be converted to a cancer causing gene by mutation
In which ways can a proto-oncogene be converted into an oncogene?
- point mutation
- gene amplification
- chromosomal translocation
- local DNA rearrangement
- insertional mutagenesis
How does the 3T3 transformation assay provide evidence for oncogene creation?
Transfection of DNA to detect non-viral oncogenes
1) transfect human tumour DNA into normal mouse fibroblasts
2) there is formation of a focus of morphologically transformed cells
3) inject into host mouse
4) tumour forms
What types of chromosomal rearrangements are there?
Deletions, translocations, duplications, inversion
What is the basis for chronic myelogenous leukaemia?
Truncation to chromosome 22, juxtaposesBCR-Abl
Transcript is constitutive,y active
What is the basis for burkitt’s lymphoma?
Translocation between chromosomes 8+4
The proto-oncogene is translocation to C14, next to the promoter. Therefore, it is expressed inappropriately due to IgH promoter proximity
What is MYC?
Transcription factor forming a heterodimer with Max, activated by mitogenic signals to trigger active proliferation. Inactivated as MYC dissociates from the complex and is replaced by Mad
What is BRAF?
A constitutively active kinase that drives high levels of cell proliferation
Raf is a serine/threonine specific protein kinase
Outline the steps of signal transduction
1) reception - hormone or growth factor binds to receptor, triggering receptor activation
2) transduction - receptor transmits signal to the inside of the cell and signal is amplified
3) response - cellular response to original signal
What are the key pathways in human cancer?
- growth factor signalling
- MAPK signalling
- PI3K/AKT
- Wnt
- hedgehog
- notch
what is the ‘input layer’ in cancer signal transduction?
ligands and receptor
what are the ‘hidden layers’ in cancer signal transduction?
adaptors and enzymes
signalling cascades
transcription factors
what is the output layer in cancer signal transduction?
changes to: apoptosis migration growth adhesion differentiation
give an example of EGFR receptor can be deregulated in cancer
v-ErbB mutant form of the protein is truncated at the CTD, making the receptor constitutively active by facilitating ligand independent firing.
what is Herceptin?
mAb that blocks ligand binding to Her2, clinically useful in patients with Her2 positive breast adenocarcinoma
outline Ras activation by RTKs
1) growth factor binds RTK
2) autodimerisation and phosphorylation of the receptor
3) phosphorylation of Grb2/Sos triggers GDP for GTP exchange on Ras
4) Ras/Raf becomes phosphorylated, phosphorylates MEK
5) phosphorylation of MEK triggers phosphorylation of ERK
6) ERK phosphorylates cytoplasmic and nuclear substrates, promoting growth, proliferation and survival
what are the three members of the Ras family?
HRAS, KRAS, NRAS
what is the functional effect of mutations on Ras?
compromised ability of GAPs to hydrolyse GTP bound by Ras, therefore, Ras remains active for longer
what is the difference between class IA and IB PI3Ks?
class IA - activated downstream of RTKs class IB - activated downstream of GPCRs
what is a class IA PI3K composed of?
p85 subunit - carries the SH2 domain that binds to the receptor
p110 (alpha, beta, omega) - kinase subunit
how are class IA PI3Ks oncogenic?
promote cell growth, survival, metabolic changes, angiogenesis, invasion and metastasis
what does PI3K do?
lipid kinase - phosphorylates 3’ position of inositol phospholipids in the plasma membrane - converting PIP2 to PIP3
what is the functional difference between PIP2 and PIP3?
PIP2 is the preferred substrate for PI3K isoforms activated by RTKs
PIP3 can activate other serine/threonine kinases as part of a signal transduction cascade
how does PTEN regulate PI3K signalling?
1) PIP3 activates PDK1 by binding PH domain
2) PDK1 phosphorylates and activates AKT/PKB
3) AKT/PKB has multiple cellular targets, regulating hallmarks of cancer
4) PTEN dephosphorylates PIP3, reversing the effects of PI3K
when is wnt signalling turned on?
during growth, tissue homeostasis and cancer
outline wnt signalling upon stimulation
extracellular wnt ligands form trimeric complexes with LRP5/6 and frizzled (receptor), activating downstream wnt pathway. Axin destroys the destruction complex, freeing beta catenin to interact with the TF -> transcription of wnt target gene
how is wnt signalling regulated by the destruction complex?
when wnt receptor complexes are not active, CK1 and GSK3a/B sequentially phosphorylate B-catenin at a series of highly conserved Ser/thr residues near its N-terminus
phosphorylated B-catenin is then recognised by B-TrCP, an E3 ubiquitin ligase, which leads to ubiquitinylation and proteasomal destruction
outline hedgehog signalling
1) hedgehog ligand binds to patched
2) patched moves away from smoothened
3) smoothened activates Gli1, which translocates to the nucleus
in which ways is hedgehog signalling commonly perturbed in cancer?
- LoF mutations to patched
- GoF mutations to smoothened
outline the notch signalling pathway
1) notch binds ligands (cell membrane bound)
2) induction of cleavage of notch between its ectodomain and transmembrane domain
3) fragment of notch is freed into the cytoplasm
4) fragment translocates to nucleus, acting as TF
how is notch signalling involved in cell-cell communication?
involved in pathways regulating stem cell development and differentiation
what are the cancer-promoting roles of Notch?
- oncogene
- tumour suppressor
- tumour progression
- survival of tumour
- drug resistance
