Session 4: Cancer Flashcards
What is a tumor suppressor gene
Tumor suppressors normally function to control cell growth and proliferation
LOF mutations contribute to the abnormal proliferation of cancer cells
What is an oncogene
- A gene that normally is involved in controlling cellular proliferation.
- When altered/over-activated, oncogenes can help transform normal cells into tumour cells by promoting uncontrolled cell growth.
Associated with GOF mutations
What are the main categories of TSG
Gate Keeper TSGs- control cell cycle progression e.g. TP53, cyclins and CDKs. e.g. mutation of TP53 (normally inhibits cell cycle progression) is mutated in 50% of tumours
Caretaker TSGs- maintain the fidelity of the genome by repairing damage e.g BRCA1/2 (HR), MSH2, MSH6, MLH1, PMS2 (MMR), MUTYH (BER) etc
How do TSGs restrain cell growth
- Inhibit the cell cycle (cyclins, CDKs, RB, TP53, APC)
- Apoptosis (TP53)
- Repair damage
Give 2 examples of TSGs
RB1
TP53
What is the Knudson hypothesis?
The Knudson hypothesis is derived from the genetic mechanisms underlying RB1 (first TSG discovered)
Explained the differences between hereditary (early onset bilateral retinoblastoma with risk of cancer in other tissues) and sporadic RB (usually unilateral, later onset).
2 hit hypothesis in which both alleles of a TSG must be lost to develop cancer. In hereditary cancer one copy of the gene is already KO so only a single additional acquired mutation is required resulting in earlier onset. this results in variable penetrate and apparently AD inheritance
Describe Retinoblastoma
early onset (<5yrs) aggressive childhood cancer of the eye. Characterised by whitening of the pupil.
Can be unilateral (usually sporadic) or bilateral (usually familial + increased risk of soft tissue and bone cancers)
Mutation spectrum includes SNVs, CNVs, SVs and hypermethylation of the RB1 promoter (10%). 60-70% display LOH of 1 allele with a mutation in the other allele. truncating mutations and deletions associated with almost complete penetrance whereas missense and splice site can have reduced penetrance and variable expressivity
What is the role of the RB1 gene
RB1 is found at 13q14
Key role in G1/S phase cell cycle checkpoint
nuclear phosphoprotein which is involved in cell cycle progression. When it is unphosphorlylated it bind E2F transcription factor preventing it entering the nucleus to activate transcription of target genes= cell cycle repression
When phosphorylated by cylcin D CDK 4/6 it dissociates from E2F which can then activate transcription of target genes (cylin E) and the cell can progress from G1 to S phase.
Describe p53’s role as a TSG
- second TSG to be discovered
- mutated in 50% of tumours
- essential in multiple signalling pathways associated with cell cycle, apoptosis and DNA repair
How does p53 contribute to cell cycle control
p53 can act as a transcription factor to activate the transcription of genes associated with cell cycle arrest and apoptosis.
In normal cells it is bound to MDM2 which retains it in the cytoplasm. (MDM2 required phosphorylation to migrate to the nucleus and bind to p53 and cause it to migrate to the cytoplasm) in the cytoplasm p53 is degraded by the ubiquitin/pretoeosom pathway.
in response to stress p53 is phosphorylated and acetylated = it can dissociate from MDM2 and activate transcription of genes e.g. PUMA which controls apoptosis.
Methods for loss of p53 function
- mutations to upstream genes e.g. ATM or CHEK2
- mutations in p53 (~50% of tumours)
p53 mutations can be LOF or GOF - mutations in gene that act downstream of p53 e.g. PTEN (germline mutations associated with Cowdens syndrome)
Describe the role of CDKN2A in the cell cycle.
CDKN2a encodes 2 unrelated proteins:
p16 INK4a- this inhibit CDK4/6 keeping RB1 dephosphorylated and bound to E2F = inhibits cell cycle
p14ARF- destabilises the interaction between MDM2 and p53 = p53 active resulting in cell cycle arrest
Germline mutations in CDKN2a is associated with malignant melanoma (penetrance depends on age and sun exposure)
Describe 2 examples of miRNAs that can function as TSGs
- Let-7
normally expressed in differentiated tissues but lost in NSCLC
negatively regulates cell cycle oncogenes and exogenous application to human lung caner cell reduces proliferation - miR-34 family
Lost in lung cancer
expression activated by p53 and associated with apoptosis
Name 3 additional TSGs, their molecular function and associated cancer susceptibility syndrome
- BRCA1/2- DSB repair and HR (HBOC)
- Lynch syndrome- MMR (CRC)
- APC- negative regulator of b-catenin. It is part of the B-catenin complex and targets it for ubiquitin mediated degradation. (FAP)
What are the 5 broad categories of oncogenes?
- secreted growth factors
- growth factor receptors
- Signal transducers - PIK3A
- Inhibitors of apoptosis - BCL2
- Transcription factors EWS-FLYI
Give an example of a secreted growth factor acting as an oncogene
secreted growth factors can act as an oncogene due to the constitutive activation of a growth factor gene e.g. normal wnt/b-catenin signalling is involved in embryonic development whereas over activation of the pathway is involved in many cancers including breast cancer.
Give an example of a growth factor receptor acting as an oncogene
e.g. EGFR and RET
EGFR receptor is constitutively expressed in NSCLC
- activating mutations occur in exons 18, 19 and 12.
- The gene encodes a RTK and mutation results in constitutive activation and over activation of downstream pathways
- Activating mutations can result in dependency for the cancerous cell on aberrant EGFR signalling
- Can be targeted by specific anti-EGFR therapies
- mutations can occur in the ATP binding pocket which reduce the affinity for ATP and increase the sensitivity to the RTK which competes with ATP for binding.
- Resistance mutations can occur in the catalytic domain which weaken the interaction between the inhibitor and its target
Give an example of a secreted signal transducer acting as an oncogene
PI3KA
- calls 1 signal transducer
- composed of a heterodimer of a catalytic and regulatory subunit and results in phosphorylation of phosphatidyl inositol lipids.
- plays a role in cell motabilism, motility and cell cycle regulation
- transmits signals from RTKs and GPCRs
- 13% have PI3KA mutations with a hotspot in the kinase domain
- RTK phosphorylates and activates PI3KA
- PI3KA phosphorlyates the inositol ring of PIP2 and converts it to PIP3
(PIP3 can also be directly activated by RAS by biding to the catalytic domain) - PIP3 is active and can directly bind and activated proteins with a pleckstrin homology domain e.g. PDPK1 and AKT which results in cell growth and proliferation.
PTEN tightly regulate PIP3 in normal cells by dephosphorylation to the inactive PIP2
Give an example of a an oncogene which inhibits apoptosis
BCL2
cytoplasmic protein which localises to the mitochondria and inhibits apoptosis.
overexpressed in most follicular lymphoma
t(14;18) BCL2-IGH rearangement results in BCL2 being under the control of the IGH locus
Give an example of a transcription factor acting as an oncogene
EWS-FLI1 rearrangement in Ewings sarcomma
- t(11;22)(q24;q12) most freq
- found in soft tissue and bone cancers
- results in a fusion between the FLI1 gene resulting in an aberrant transcription factor = aberrant regulation of growth and cell proliferation
What are the main mutation mechanisms for activating an oncogene (GOF)
- Point mutations- can constiutively activate a signalling pathway e.g. mutation to the regulatory domain or dimerisation domain. Results in hyperactivation of a protein that is expressed in normal amounts
- Amplification- overexpression of a non-mutated gene leading to excess protein e.g. Her-2 in BC or MYCN in NB
- Translocation to produce a novel fusion gene- aberrant or dysregulated function. e.g. BCR-ABL in CML
- Translocation into a transcriptionally active region e.g. Burkitts Lymphoma. t(8;14)(q24;q32) is seen in 75% of patients and results in juxtaposition of the MYC oncogene with an immunoglobulin (IG) locus and consequently, MYC is brought under transcriptional control of the IG locus at the same time as losing its own. Other Ig locus rearrangements are also common in cancer.
- Local DNA rearangements- fusion genes can be created by inversion or deletion of the intervening region between a gene e.g. Inv(16) in AML
- Insertional mutagenesis- Hepatitis B in hepatocellular carcinoma. Viral oncogenes insert near cellular genes such as MYC and aberrantly activate it to initiate unchecked cellular proliferation.
what is tumour mutation burden (TMB) testing?
it is a measurement of the total number of non synonymous mutations in the tumour exome
the number of somatic mutations per megabase of interrogated genomic sequence, varies across malignancies.
What is the clinical utility of TMB?
can be used as a biomarker for immunotherapy, especially for the use of immune checkpoint inhibitors.
how does TMB influence immune checkpoint inhibitors?
somatic mutations can result in the expression of neoantigens and the chance is greater the higher the TMB
The neoantigens can activate the proliferation of T-cells which act to kill the cancer cells and such tumours can be targeted with immune checkpoint inhibitors-
How do checkpoint inhibitors work
these work by blocking the binding of checkpoint proteins to their partners
prevent the off signal from being sent so T-cells kill the cancer cells.
ICIs show variable efficicy and biomarkers help stratfiy patients that will respond
- MSI high tumours also show good response to ICIs as there is a high TMB
high TMB correlates with a good response to ICIs and increased survival in some cancers including NSCLC
response is not consistent across different cancer types and there is no established threshold or methodology for TMB
What tests can be used to determine TMB?
WES/WGS panels- WES considered best but not as cheap or fast as targeted panels- targeted panels are used to exptrapolate the number of somatic coding mutations observed in the targeted genomic space to the no. that would be observed across the whole genome. tumour percentage, qualit and seq depth all influence the number of somatic mutations detected.
standardization is required to fully assess and implement TMB as a biomarker in the UK
what does ‘actionable’ mean in terms of variant interp
variant can be used a biomarker for the patients disease
what is a biomarker?
a marker of disease that can provide info that is useful for the diagnosis, prognosis or treatment of a patient
What are the current UL guidelines for somatic variant interpretation
ACMG and ACGS BPG for variant classification state that a different set of interpretation guidelines is needed for somatic variants
2017 American association for molecular pathology released guidance and a working group has been established in the UK
what are the differences between somatic and germline variant interpretation
constitutional-
homogeneous, allele fraction 0.5 or 1 (unless mosaic), low AF can generally be discounted (be wary of mosaic),
family studies are possible
Is the variant causative of phenotype?
germline- tumour heterogeneity
variable allele fraction
low AF- genuine or seq artifact
cannot use linkage
Is the variant a driver of tumourigenesis, useful for tumour classification, have prognostic implications, target-able with a drug?
describe the difference stages of the cells cycle
G1= growth 1. RNA and proteins are synthesised (not DNA). each chromosome exits as a double helix
S- DNA synthesis- each chromosome is now present as sister chromatids
G2- cell continues to grow
M- mitosis- cell stops growing and there is nuclear division (miosis) followed by cellular division (cytokinesis) to produce to daughter cells.
Go= senescence the cell has left the cell cycle and stopped dividing. the cell may re-join the cell cycle in response to specific signals.
How is the cell cycle controlled?
cell cycle checkpoints
what are the cell cycle checkpoints?
- G1/S checkpoint (restirction point) once passed the cell is commited to division and enters S phase
- G2 checkpoint ensure there is enough cytoplasmic materials available for mitosis and cyotkinesis
- Metaphase checkpoint at the end of mitosis ensure that chromosomes are correctly attached to the spindle.
What controls the cell cycle checkpoints
controlled by heterodimeric protein kinases.
Consist of:
- constituitively expressed cdk kinase- this provides the catalytic unit but is inactive without its cyclin parter
- cyclin regulatory unit which is expressed at specific times in the cell cycle.
Together they act to phosphorylate target protein and their action is reversed Cdk inhibitors (CKI’s) = phosphatases. CKI’s are frequesntly mutated in cancer.
describe the control of the G1 restriction point.
after this checkpoint the cell is irreversibly committed to cell division.
controlled by cdk4/6-cyclin D complex whihc is formed in response to growth
cdk4/6-cyclin d phosphorylation and inactivates RB1 (TSG), releasing its inhibition of E2F (transcription factor). E2F can enter the nucleus and activate transcription of genes involved in cell cycle progression.= cyclin E is produced and forms a complex with cdk2 results in in G1/s transition.
the RB1 is hypophosphorylated it is in its active form and it bound to E2F, inhibiting its TF action and preventing cell cycle progression.
Give examples of disregulation of G1/S checkpoint in cancer
In retinoblastoma RB1 is mutated so that it cannot bind E2F and control cell cycle, resulting in uncontrolled cell growth
Cyclin D is overexpressed in many cancers resulting is phosphorylation of RB1 and loss of its cell cycle control even in the absence of growth signals. Cyclin D over expression can be induce by the PI3K and RAS pathways.
how can TP53 control the cell cycle?
- can arrest the cell in G1 by the production of CKIs (reverse phsphorylation by Cdks)
- can trigger apoptosis or production of DNA repair enzymes by acting as a TF
How is TP53 regulated at the G1/S checkpoint?
in normal cells TP53 levels are kept low by binding to MDM2. TP53 induces MDM2 expression resulting in a negative feedback loop
- MDM2 is a ubiquitin ligsae that must be phosphorylated to migrate to the nucleus and interact with P53.
MDM2 binding to TP53 sequesters it in the cytoplasm where it is ubiqutinated and targeted to the proteosome for degredation.
in response to stress TP53 dissociates from MDM2. this allows it to travel to the nucleus and activate transcription of genes involved in cell cycle arrest and apoptosis e.g. PUMA
How does CDKN2A contribute to cell cycle control?
Transcribed into two unrelated genes
1. p16INK4A- inhibits cdk4/6 so RB1 is not phosphorylated and remains bound to E2F preventing the transcription of cyclins required for cell cycle progression
2. p14ARF destabilizes the MDM2-P53 interaction so p53 is released and can activated cell cycle arrest in G1
CDKN2A is mutated in many cancer we.g. multiple melanoma. cancer risk is dependent on age and sun exposure
How is the G2/M checkpoint controlled?
Cdk1 is activated by phosphorylation and dephosphorylation of specific residues and the formation of the Cdk1-cyclin B complex (AKA MPF)
How is the metaphase (spindle checkpoint) controlled?
The spindle checkpoint ensures that the chromosomes are correctly aligned on the metaphase plate and that sister chromatids are correctly attached to the spindle.
The anapahse promoting complex is activated -> this degrade cyclin B (MPF disassembly) which releases inhibition from seperase resulting in sister chromatid separation and progression to anaphase
How does disregulation of the cell cycle contribute to tumorgenesis?
failure to activate checkpoint in response to damage results in maintenance of the damage and genome instability- mutated or damaged cells remain in the cell cycle and or there is increased cell proliferation.
what are possible cell cycle therapeutic targets?
- gene silencing has been used to target MDM2- this prevents its interaction with TP53, allowing levels to rise in the cell and activation of cell cycle arrest and apoptosis.
- can inhibit the E3 ubiqutin ligase activity of MDM2 o prevent it from targeting TP53 for degredation.
how does meiosis differ from mitosis?
there are 2 rounds of division in meiosis resulting in the production of 4 haploid daughter cells.
what are the phases of meiosis?
Prophase 1
mat and pat homologues form bivalents and are held together by the synaptonemal complex. the synapsed chromosomes undergo recombination. The recombining chromosomes are physically connected at the location of the crossover (chiasmata)
chiasmata are important for correct separation in anaphase
Metaphase 1
spindle is formed and bivalents align on the metaphase plate
Anaphase 1
homologues separate. chromatids remain attached
Telophase 1
chromosome are separated at each pole and daughter cells form
Prophase/metaphase 2
cells pass directly from meiosis 1 to metaphase II with no prophase. The nuclear envelope break down, a new spindle forms and the chromosomes align (2 chromatids)
Anaphase 2
separation of centromeres and migration of chromatids to opposite poles
Telophase 2
further cell disvision occurs forming 2 haploid daughter cells.
Describe the importance of damage repair in cancer
Damage repair is important to maintain genome stability
Damage from exogenous and endogenous agents is repaired and mismatches introduced during DNA rep
the processivity of the DNA pol is the rate limiting factor in determining the mutation rate
Describe the role of NER
Nuceotide excision repair
Reparis pyrimidine dimers caused by UV damage or large chemical adducts
1) NER machinery (complex of >30 proteins) recognizes the damage as it disrupts the DNA helix.
2) nuclease excision so ther damaged DNA and surrounding bases
3) gap fill by a DNA pol using the complimentary strand as a template
4) nick ligated
there are 2 classes:
- global excision repair (repairs all DNA)
- Transcription repair (repairs DNA undergoing transcription)
defective in Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy - increased sun sensitivity.
How does DNA damage trigger repair or apoptosis?
DNA damage activates cell cycle checkpoints resulting in cell cycle arrest and apoptosis.
failure to activate check point results in an accumulation of damage and and can result in oncogenesis.
ATM and ATR are checkpoint regulators and their dysfunction is associated with increased cancer susceptibility
Describe mismatch repair
Repairs mismatches, insertions and deletion arising during DNA replication.
- MutSa (MSH2 + MSH6) recognises mismatches and small indels
- MutSb (MHS2 + MHh3) recognises larger looped out ins and dels
MutSa recognises damaged DNA and recruits MutL (MLH1 and PMS2)- this results in excision of the mismatched bases, there is gap fill by a DNA pol and the nick is ligated by a ligase.
Mutations in MMR protains are associated with Lynch syndrome
Describe base excision repair
BER excises non-helix distorting lesions form the DNA- e.g. damage from oxidation of alkylation.
glycolases cleave the glycosidic bond between the sugar and the damaged bases to leave an AP (apurinic or apyrimidinic) site.
e.g. MUTYH (associated with AR MAP) cleaves the glycosific bond to remove 8-oxo-G caused by oxidatuve damage. If un-repaired this pairs with T instead of C resulting in a GC to AT transition.
A phosphatase then cleaves the phosphodiester bond 5’ to the AP site leaving a 5’ sugar phosphate and 3’OH. the sugar phosphate is removed to leave a single nucleotide gap which is filled by a polymerase and the DNA is ligated.
Describe NHEJ
non replicative mechanism
error prone and is responsible for many of the recurrent rearrangement seen in cancer. (50% of recurrent rearrangements have 1 breakpoint in a fragile site)
Repair is by end-joining of 2 DSbs with no homology required. Often results in small insertions or deletions at the breakpoint. The ends are edited to reveal microhomologies
1) DSB is recognised by the Ku 70/80 heterodimer which forms a scaffold that holds the DNA ends together- after formation of a synapse in whihc the broken ends overlap the KU heterodimer unwinds a small part of the DNA to reveal a region of microhomology that is present by chance
2) Artemis: DNA dependent protein kinase with 2 functions
- exonuclease to trim excess unparied overhanging ss ends
- exonuclease activity that cleaves haripins together
3) DNA pol fills remainin gaps
4) end joing by DNA ligase
NHEJ-LIGiV and XLF-SCID are 2 syndromes that are associated with dysfunction in NHEJ
Describe HR
- High fidelity repair mechanism that uses the sister chromatid to guide repair in G-phase.
- requires extensive homology (>300bp)
RAD51 is the strand exchange protein required to catalyse the invasion of the homologous DNA sequence– mediates ss invasion of the homologous sequence by the 3’ end of the ss DNA to replace the equivalent strand.
BRCA1 and BRCA2 also required to co-localise with RAD51 at the site of DNA damage to activate the repair.
NBS and BLM (helicase) are also involved in HR.
BLM is associated with Bloom syndrome (sunsensitivity, butterfly shaped red mark on face and increased risk of cancer).
NBS is associated with Nijmegen breakage syndrome (microcephaly, facial dysmorphism and increased cancer risk)
Describe translesion synthesis
Major source of mutations
Uses a low fidelity polymerase which can replicate damaged DNA and bypass a stalled replication fork but this is at the cost of a high error rate.
- low fidelity pol often incorporates mismatches even in undamaged DNA
the high error rate can be advantageous and contribute to diversity of immunoglobulins.
paradoxically translesion synthesis plays a role in suppression of cancer. It can fill passed replicative gaps and suppress the DNA damage response including cell cycle checkpoints. cellular senescence and apoptosis
- also supresses genomic rearamngements and stalled forks do not turn into DSBs.
Give examples of the cancer susceptibility syndromes
Fanconi anemia
Ataxia Telengiecstasoia
Xeroderma pigmentosum
Nimejhan breakage syndrome
Li fraumeni
HNPCC
HBOC
MAP
Give examples of the repair mechanisms and tumour susceptibility for the cancer susceptibility syndromes
FA- FA genes, corss link repair,m increased risk of AML
AT- ATM gene, DSBs and checkpoints, increased risk of acute leukemias and lymphomas
XP- XP genes, NER, increased risk of skin cancers
NBS, NBS gene, DSBs and checkpoints, increased risk of acute leukemias and lymphomas
Li Farumeni, TP53, apoptosis and checkpoints, mutated in 50% of tumours
HNPCC, MMR genes, increased risk of CRC
HBOC- BRCA1/2, HR, increased risk of breast and ovarian cancer
MAP, MUTYH (AR), involved in BER, increased risk of MAP
Describe synthetic lethality in cancer therapy?
Synthetic lethality can be used to target DNA repair pathways. It i based on the phenomenon that the presence of 2 genetic events in related pathways results in apoptosis in the presence of other stresses.
- it indicates a relatedness between 2 gene
- can be considered a feature of genetic robustness- mechanism for maintaining genome stability in the presence of stress. There is some redundancy in different pathways meaning that it one is defective an other can compensate. However loss of both will result in cell death. This can be used to selectively target cancer cells which have already mutated one of the pathways (e.g, HR in HBOC) as part of their oncogenesis. Normal cells will not be affected as they still have a fully functioning original pathway (HR)
Give an example of synthetic lethality in cancer treatment
E.g. BRCA mutations in HBOC show synthetic lethality with PARP inhibitors
-PARP inhibitors are DNA repair proteins involved in ssBs repair
- ssBs cant be repaired so are converted to DSBs at rep fork. These are also not repaired resulting in apoptosis due to the accumulation of a high level of DNA damage
- normal cells still have functioning BRCA so can repair the lesions
How is synthetic lethality identified?
you can investigate the drugability of SL targets in DNA damage response pathways using SL screens…. using genetic variability of cancerous cell or genetically engineered KO cells and investigating the functional consequences
-RNA1 screening have been used to investigate SL associated with RAS
What is stratified and personalised medicine? what are the benefits?
Using biological markers e.g. the presence of specific genetic markers to straify patients into groups based on their response to treatments
benefits:
-safer and fewer ADRs
- better response to treatment
- improved mores specific diagnostics
-only treat those that will benefit reducing costs
- improved choice
Given an example of stratified medicine in breast cancer
ERBB2/HER-2 overexpression
ERBB” encodes the growth factor receptor tyrosine kinase HER-2
- this is overexpressed in 20-30% breast cancers (Her-2 +ve) and is associated with a mores agressive cancer and high recurrence risk. (Unlikely in BRCA1 which is associated with triple -ve breast cancer)
It can be treated by the drug herceptin. Herceptins side effects include cardiac disease so only want to treat patients that will benefit.
How is HER-2 overexpression tested for?
HER-s expression is detected by IHC of tumor samples. This testing is recommended by the UK cancer network guidelines for all primary or metastatic breast cancers
staining is rated as:
0-1+ = no staining or incomplete membrane staining. Less than 10% of tumours with this level of staining will have HER-2 over expression
2+= weak to moderate membrane staining ~10% of tumours with this staining will have HER-2 over expression, re-test.
3+ = complete membrane staining. HER-2 +ve
often recommended to re-test IHC (measures protein expression) with FISH (measures gene expression)
Herceptin resistance is an issue that may result from activating PI3K mutations or inactivating PTEN mutations
Given an example of stratified medicine in hereditary breast cancer
BRCA1/2 mutations are synthetic lethal with PAR inhibitors e.g. olaparib
PAR inhibitors have been shown to stop or shrink the growth of breast, ovarian and prostrate tumours.
what are the most common genetic changes in lung cancer?
the most common mutations in lung cancer are KRAS and EGFR
ALK rearrangements resulting in aberrant fusion proteins are also common. Mostly EML4-ALK
Describe stratified medicine for EGFR in lung cancer
EGFR activating mutations are common in NSCLC and is associated with a better prognosis
EGFR is a TM tyrosine kinase receptor involved in cell growth and proliferation. Activating mutations result in constitutive downstream signalling and uncontrolled cell growth.
- mutations are found in the tyr kinase domain (exons 18-20)
EGFR positive lung cancer can be treated by Gefitinib. The drug is 100x more effective in EGFR +ve lung cancers. It works as a TKI by blocking the ATP binding site.
