Cancer genomics

Question 1

What are the 2 next gene sequencing technologies likely to come into mainstream use?

Answer 1

Oxford Nanopore
Pacbio
Both are too expensive atm (but will get competitive soon). They can read up to 10kb but make mistakes. This is in contrast to Illumina which can read 150 bases but is more accurate. Long reads with mistakes would help string sequences together.

Question 2

How does exome sequencing work?

Answer 2

Break up genomic DNA, hybridise with bits you’re interested (e.g. exome) and pull down. Sequence these bits. Lose some information but is cheaper.

Question 3

How does Illumina sequencing work?

Answer 3

Error rate is 1/100 or 1/1000 depending on location in genome. Do paired end sequencing (sequence from both ends) of 250-500bp fragments of DNA. Get approx. 150bp in from each end. Then align to a reference.

Question 4

What are the problems with sequence alignment in Illumina?

Answer 4

If there are too many repeats/errors in the sequence it can’t be lined up
If there are many mutations it won’t align perfectly - go for where it is closest but may be in wrong place or won’t align at all.
If there are translocations, they will be found if one end of the fragment aligns with chromosome a and the other aligns with chromosome b. Have to allow for mismatches however as there is a high error rate in this process - misalignment (where there is homology in reference genome on another chromosome) is as common as a translocation. Can also get translocations when repairing fragments if they join with a piece from another chromosome.
LOTS of noise in translocations, need to sequence many fragments

Question 5

How can misalignments in Illumina sequencing be verified?

Answer 5

Can’t do by Sanger as is too expensive. Look at agreement between labs using the same data – 80-90% agreement for single base mutations; 30-40% for INDELs. Due to use of different software.
Artefacts of the sequencing can lead to findings of ‘translocations’ in cancer which are false.

Question 6

How can cancer mutations be found by non-genome methods?

Answer 6

Historically looked in retroviruses that cause tumours in animals. If retroviruses are selected for efficiency, many had acquired an oncogene which could be confirmed by sequencing the virus
Can transfect oncogenes into cells and look for phenotype

Question 7

How can cancer mutations be found by genome-wide methods?

Answer 7

Hereditary predisposition (map, find location, find tumour w/ deletion and sequence) - helped find tumour suppressors
Cytogenetics (oncogenes in translocations)
Losses by cytogenetics and loss of heterozygosity (looking for common deletions in cancer)
CGH arrays
Sanger sequencing screens (e.g. MAPK screen found BRAF)

Question 8

What are the advantages and disadvantages of using cytogenetics to look for chromosome translocations?

Answer 8

Good in leukaemia as there are few translocations
Not good in epithelial cancers where there are too many translocations. Improved a bit with FISH but still don’t know what parts of the chromomse have swapped so can’t tell significance.
Also can’t pick up small translocations e.g. TMPRSS2-ERG - TMPRSS2 gives promoter, ERG is a transcription factor. Is a small gene fusion

Question 9

What has CGH arrays taught us about cancer genomics?

Answer 9

Comparative genome hybridisation arrays. Able to count the number of copies of a genome region in cancer (deletions and amplifications) - informs about copy number variants. Normal DNA is dyed green, tumour DNA is dyed red, they are hybridised and a signal is looked for. Doesn’t take to gene level - looking at 100kb areas at best. Has been superseded by whole genome sequencing - count reads at each location.

Question 10

What have Sanger sequencing screens taught us about cancer genomics?

Answer 10

PCR exon by exon and sequence and look for statistically significant results. Can look for candidate screens e.g. hypothesis beta-catenin could be an oncogene and go look for mutations. Also, screens for the MAPK pathway have been done.

Question 11

What is the significance of TTC28?

Answer 11

Has a mobile element in an intron and therefore is translocated all over the genome, with the same sequence with in 4kb. This is found a lot in cancers as non-LTR LINEs can be activated. LINE is copied and takes part of TTC28 with it.

Question 12

How does LINE1 move around the genome?

Answer 12

mRNA is transcribed and translated to make reverse transcriptase. Get DNA copied from the mRNA. In the case of TTC28, the mRNA extends into TTC28 and is therefore carried when it is reverse transcribed. L1 is polymorphic and not unique so can’t align and is therefore not detected.

Question 13

What is the current knowledge of cancer genetics?

Answer 13

Point mutations in exons - high reliability, 50% sensitivity
Indels in exons - less than 50% reliability
Small mutations in non-coding sequences (promoters, enhancers) - little known
Structural rearrangements - few from sequencing, well known ones for cytogenetics/CGH
Mobile element insertions - only just discovered
Epigenetics - poor

Question 14

What are the different levels of transcription regulation?

Answer 14

DNA state (histone code, DNA methylation, chromatin structure)
Transcription factors
Co factors
Protein post translational modifications
RNA Pol II mediated gene expression (near promoters)

Question 15

Describe the histone modifications found at enhancers and promoters?

Answer 15

Are distinct from each other
Enhancers: mono/dimethylation of H3K4 (not tri, required for enhancer function)
Promoters: trimethylation of H3K4 for activation. Also acetylation. Methylation at K9 and K27 is repressive.

Question 16

How can enhancers be identified?

Answer 16

Tend to be in euchromatin. Can be identified using DNase I hypersensitivity. Embryonic stem cells have ~3% of the genome open (could be an enhancer). These close as cells differentiate.

Question 17

What is special about nuclear receptors?

Answer 17

Are the only transcription factors that can be constituently switched on by a ligand.

Question 18

Describe the structure of nuclear receptors

Answer 18

Have a variable section at the N terminus, then a DNA binding domain consisting of 2 zinc finger domains, then a ligand binding domain.

Question 19

How can nuclear receptors be drugged?

Answer 19

The ligand binding domain is unique and therefore druggable

Question 20

How do nuclear receptors act in cancer?

Answer 20

Oestrogen-ER causes breast cancer (75%)
Androgen-AR causes prostate cancer (100%)
Are key targets in therapy and provide models to learn about transcription and cell growth.

Question 21

How do cofactors work with nuclear receptors?

Answer 21

A very complex interaction involving many co-factors. Varies between cell type - not all co-factors function in all cells. Can edit histone modifications e.g. CBBP/p300 is tethered to the nuclear receptor by SRC-1 and others and they can acetylate histones. Cofactors are commonly mutated in cancer.

Question 22

How can the binding points for ER be found in an unbiased way?

Answer 22

Use Illumina to find the DNA bound to the transcription factor. Pull down ER and sequence, remove noise and look. Is an unbiased method that is reproducible. Found that most binding sites were in the middle of nowhere.

Question 23

What did the discovery of where ER binds tell us?

Answer 23

Found that ER mostly bound in the middle of nowhere. Thought to not be random as DNA was DNase I hypersensitive and had histone markers (H3K4me1/2). Also seemed to have a cis regulatory element (the oestrogen responsive element or the forkhead TF binding motif. for a forkhead protein).

Question 24

What is FoxA1?

Answer 24

Is a forkhead box protein. Is a pioneer factor that binds forkhead motifs at tightly wound DNA (not heterochromatin) and opens it up to allow other transcription factors to bind.

Question 25

How was it found which ER binding site activated which gene?

Answer 25

Hypothesised chromatin looping. Did chromosome conformation capture and ChIA-PET - fix with formaldehyde, cut with restriction enzymes and religate. Then pull down and sequence. Found flexible loops covering the whole genome, but no inter chromosome interactions. Found that anchor genes are brought to a hub of ER and FoxA1 and those looped out are silenced. TFF1 and TFF2 are activated by oestrogen and ER binds the promoter (one of the only examples)

Question 26

What is GRO-seq?

Answer 26

Take all transcriptions, biotinylate and pull down then sequence. Is an unbiased way of looking at all transcripts and found that intervening DNA between genes is super active

Question 27

How did GRO-seq inform us about ER activity?

Answer 27

27% of whole genome is transcribed when ER is active. Includes enhancer RNAs along with ncRNAs, antisense transcripts, divergent transcripts etc

Question 28

What do eRNAs do?

Answer 28

Thought to aid chromatin looping - forming bridges between loops. If they are silenced get loss of cell growth and abnormal gene expression.

Question 29

How is the TMPRSS2-ERG fusion associated with nuclear receptors?

Answer 29

TMPRSS2 is a gene controlled by the androgen receptor. When this fusion takes place, the TMPRSS2 promoter is fused to an oncogenic transcription factor (ERG) and expression is activated by androgen. There are also other examples of fusions of ETS factor and AR target genes

Question 30

How can DNA properties be drugged in cancer?

Answer 30

5-aza-cytodine is a DNA demethylation agent (need to control dosage) G-quadruplexes may be able to be targeted by inhibitors

Question 31

How can transcription/pioneer factors be drugged in cancer?

Answer 31

Could regulate other transcription factors as a decoy or block protein-protein interfaces (hard to get into the cell)

Question 32

How can co-factors and associated proteins that are mutated in cancer be drugged?

Answer 32

AIB1 is a breast co-factor where a drug has been found | IBET inhibit BRD4 co-factors

Question 33

What factors increase the risk of breast cancer?

Answer 33

Family history Factors that increase oestrogen exposure More periods in your life (early menarche, late menopause) No children (lowers oestrogen) Hormone replacement therapy High BMI (oestrogen metabolised from fat)

Question 34

What is the normal function for nuclear receptors?

Answer 34

Required for tissue development e.g. ER mediates cell growth for mammary gland development, AR mediates cell growth for prostate development, glucocorticoids, progesterone, prolactin etc are all important for normal physiology.

Question 35

How does ER cause cell growth in breast cancer?

Answer 35

Oestrogen diffuses in and binds ER dimers in the cytoplasm. These then translocate to the nucleus where they bind DNA and up regulate transcription. AR works in exactly the same way for prostate cancer

Question 36

How does Tamoxifen affect ER activity?

Answer 36

Can bind ER dimers instead of oestrogen and promote translocation into the nucleus. ER dimers bind DNA but transcription isn't up regulated (is not active.

Question 37

How does aromatase affect ER activity?

Answer 37

Oestrogen is metabolised from precursors by aromatase. If aromatase is inhibited, it limits the amount of oestrogen that can form (increases hot flushes etc)

Question 38

How does anti androgens affect AR activity?

Answer 38

Bind AR dimers in the cytoplasm and allow translation to nucleus. AR dimers sit on DNA but don't up regulate transcription - are inactive. Examples of new effective agents are abiraterone, enzalutamide.

Question 39

What did we learn from mapping AR binding sites?

Answer 39

Loads of AR binding events, like ER, in prostate cancer cells; very few promoters bound. Forkhead motifs were also enriched GATA motifs were enriched

Question 40

How does blocking FoxA1 binding affect ER binding?

Answer 40

No ER binding at all; no gene expression, no cell growth

Question 41

How does blocking FoxA1 binding affect AR binding?

Answer 41

No AR binding at the original sites, but AR goes and binds new sites. Cell still stops growing, so the movement has no effect. FoxA1 and FoxA2 is present in prostate (only FoxA1 in breast)

Question 42

How is FoxA1 associated with breast cancer outcome?

Answer 42

FoxA1 is found in all ER+ breast cancers. Can be found in all primary tumours and in metastases. ER can move around the genome, dictated by FoxA1. This has an influence on cell fate and survival rate.

Question 43

How does drug resistance arise in ER brest cancer?

Answer 43

Phosphorylation of ER, ER mutations to prevent drug binding (in ligand binding domain). Often found in metastasis.

Question 44

Why is FoxA1 a good drug target?

Answer 44

Is absolutely necessary for ER+ cancers, even in drug resistant cells. Also needed in AR+ cancers and castrate resistant prostate cancer.

Question 45

Can we drug FoxA1?

Answer 45

Maybe. Has a restricted tissue expression profile

Question 46

How do prostate cancers become drug resistant?

Answer 46

Select splicing variants with different ligand binding

Question 47

What are molecular apocrine breast cancers?

Answer 47

Are ER negative but have other hallmarks of ER positive cancers. AR can substitute for ER. Have different peaks in a Chip-seq to prostate cancer, but is very similar to ER breast cancer. AR can substitute for ER due to FoxA1 which dictates binding points - AR can go to ER binding points. Binding points have a higher affinity for ER, but if there is no ER get AR to drive genes. AR is in all ER+ breast cancers but silenced (unless no ER)

Question 48

How can molecular apocrine cancers be treated?

Answer 48

If women don't respond to tamoxifen, can give same drugs as prostate cancer which work well. However, if give these drugs to an ER+ cancer, it makes the cancer worse.

Question 49

How does progesterone interact with the ER pathway?

Answer 49

PR blocks ER binding and activity, leading to 'good' transcriptional outcomes. 20% of ER+ cancers lose PR. This occurs through a strong interaction of PR with ER and sequestering of ER. Suggested that progesterone is protective - strong correlation between levels of progesterone and age of diagnosis (as decrease get increase).

Question 50

What is a new way of treating ER+ cancers?

Answer 50

Giving progestins with ER inhibitors such as tamoxifen seems to be a potent way of treating cancer. This is in clinical trails now.

Question 51

What is the problem with treating cancers with progestins?

Answer 51

In mice, found that progestins encouraged proliferation in mouse mammary glands. This was extrapolated to say that progestins are pro-proliferative in human cancers when in fact the opposite is true. Also have data from HRT which showed that progestins gave a small increase in proliferation - due to a slight lack of specificity to PR (hits other nuclear receptors too). In France, use of progesterone (as opposed to progestins) has given protection from cancer

Question 52

What machineries modify chromatin?

Answer 52

DNA methylation Histone modification/histone variants ncRNA and relation with expression (can interact with transcription factors)

Question 53

What is EZH2?

Answer 53

Part of the polychrome repressive complex 2 (PRC2) which works to silence gene expression. Is an enzyme with a SET domain that adds methyl groups to H2K27 (up to 3 methylations) which switches off transcription.

Question 54

How can EZH2 be mutated in cancer?

Answer 54

In non-Hodgkin lymphoma: In SET domain, Y641A creates more di/trimethylation. Therefore is an attractive target for drugs - GSK126 inhibits growth of cancers with this mutation. Also get inactivating mutations in some cancers e.t. T-cell acute lymphoblastic leukaemia

Question 55

How does 5' methylation of cytosine alter gene expression?

Answer 55

Can recruit CpG DNA binding proteins Could block DNA binding proteins Could change DNA charge and alter the architecture

Question 56

How is DNA methylation affected in cancers?

Answer 56

Disturbed in 75% of cancers. Global hypomethylation with some hypermethylation at specific loci to silence certain genes e.g. TP53.

Question 57

How does epigenetics have an effect on genomic instability?

Answer 57

Changes in DNA methylation gives genomic instability, potentially due to recombination or lack of repression of transposable elements

Question 58

Where is DNA hypomethylated in cancer?

Answer 58

Oncogenes, DNA satellites, repetitive elements.

Question 59

How can DNA methylation be used as a diagnostic tool?

Answer 59

Can be used to predict prognosis. Observe hypomethylation of LINE elements and can be used to guide drug targets by looking at methylation of certain genes. Can see DNA hypomethylation in bodily fluids - could be used to diagnose cancers

Question 60

What external factors influence DNA methylation?

Answer 60

Age - genome tends to become hypomethylated but CpG islands are hypermethlated (same as in cancer, could be linked to increase cancer incidence) Diet - S-adenosyl methione is substrate for methylation, gained through folate and methionine pathway Environment - arsenic (hypomethylates Ras gene) and cadmium (global hypomethylation by DNMT1 inactivation) associated with cancer

Question 61

How can DNA methylation be detected?

Answer 61

Bisulphide addition. Convert mC to uracil, sequence and compare. Can't distinguish between mC and hmC which is part of the removal mechanism - could influence how correct the literature is

Question 62

How is DNA methylation involved in ovarian cancer?

Answer 62

Hypermethylation of tumour suppressor genes e.g. those involved in DNA mismatch repair (e.g. BRCA1, hMLH1) Hypomethylation of LINE elements (correlates with advanced cancer) Hypomethylation of TUBB3 - a determinant of taxane resistance

Question 63

What histone modifications are associated with cancer?

Answer 63

H3K4me2/3 - activating | H3K27me3 at silenced genes

Question 64

How does EZH2 expression affect expression of E cadherin?

Answer 64

EZH2 is super active in cancers and adds the histone modification H3K27 to silence genes. This occurs at E cadherin. E cadherin normally mediate cell-cell adhesion and keeps cells together and prevents moving. If it is inhibited, this allows metastasis. Found increase in cell movement in a cell invasion assay when EZH2 was over expressed and E-cadherin was silenced.

Question 65

How are BET proteins involved inn cancer?

Answer 65

Bind acetylation on histones, involved activation and transcription. In rare leukaemia's, BET proteins are misplaced and get aberrant activation

Question 66

How can BET proteins be inhibited?

Answer 66

JQ1 is a drug which binds in the pocket to block target binding. However has a short half life and causes male sterility. I-BET762 is more promising and can also be used in solid tumours

Question 67

What is Vorinostat?

Answer 67

A drug against HDACs. Works on the cancer stem cell hypothesis - the cancer transcriptome is rewritten to look like a stem cell. In aggressive cancers get a shift in gene expression profile along with change of DNA methylation and histone markers.

Question 68

What is GSKJ4?

Answer 68

A drug which restores methylation at H3K27. Used to treat diffuse intrinsic pontine glioma in which there is hypomethylation of H3K27 leading to a more accessible chromatin state and aberrant gene expression. Treatment with GSKJ4 restores methylation and causes tumour shrinkage.

Question 69

How do mutations of ncRNAs affect cancer?

Answer 69

Deletion of 13q14 region encodes miR-15/16 which regulate apoptosis. Found in chronic lymphocytic leukaemia Amplification of miR-17-92 found in diffuse large B cell lymphoma patients CNV in some miRNAs confers chemo/radioresistance Also have epigenetic control and processing defects

Question 70

What does mi-R10b do in cancer?

Answer 70

Is involved in breast cancer metastasis

Question 71

What does miR-31 do in cancer?

Answer 71

Deregulated in breast cancer, get leakage into the blood stream. Could be used as a diagnostic marker and prognosis

Question 72

What is the difference between siRNAs and miRNAs?

Answer 72

siRNAs need a perfect match (1 gene target) miRNAs don't need a perfect match and can therefore have many gene targets and wide spread effects. May have a seed sequence.

Question 73

What does miR-132 do in cancer?

Answer 73

Promoter of miR-132 is hypermethylated in prostate cancer. Silencing of miR-132 leads to increased cell death and increased invasion and migration. Targets HB-EGF and TALIN2. Is silenced by EZH2 in ovarian cancer. Also get a metabolic switch and increased GLUT1 expression when silenced.

Question 74

What is HOTAIR?

Answer 74

A long non coding RNA. Interacts with PRC2; is necessary for PRC2 occupancy at H3K27me3 at the HoxD locus and MYC in breast cancer.

Question 75

What is MEG3?

Answer 75

A lncRNA. Stimulates interaction between JARID2 and PRC2 component EZH2 leading to recruitment of EZH2 to chromatin at the imprinted locus Dlk1-Dio3 and silencing. Is a tumour suppressor - is associated with MDM2 expression and p53 accumulation.

Question 76

What is MALAT1?

Answer 76

A lncRNA which can act as an oncogene. Is associated with metastasis in lung cancer and angiogenesis. Is up regulated indirectly through N-myc.

Question 77

What is miR-101?

Answer 77

A microRNA that inhibits expression and function of EZH2. Expression of miR-101 decreases during cancer progression as EZH2 activity increases.

Question 78

What is miR-31?

Answer 78

An miRNA which is repressed through EZH2 activity. Is lost in adult T cell leukaemia. Is lost n prostate cancer through promoter hypermethylation.

Question 79

What are germ cell tumours (GCTs)?

Answer 79

Tumours that arise from primordial germ cells. Include testicular, ovarian, CNS and other locations.

Question 80

Describe the histological heterogeneity of GCTs

Answer 80

Many different histologies, due to different pathways down the differentiation lines.

Question 81

What miRNAs are unregulated in GCTs?

Answer 81

miR371-373 and miR-302/367. Found in both childhood and adult tumours. Have identical seed regions (nucleotides 2-7 which determine mRNA binding - rest don't have to be perfectly complimentary), so down regulate similar targets.

Question 82

How can miRNAs be identified as passengers or drivers?

Answer 82

Look for a seed region - can look for in down regulated miRNAs then see if it is present in mRNAs that are down regulated. See what the mRNAs code for - signalling/biological processes. In mouse embryos, high level of miRNAs are found, including miRNAs found in cancer. These are down regulated during differentiation as they can be oncogenic

Question 83

How can miRNA be used as a biomarker for GCTs?

Answer 83

At the moment use levels of serum proteins - different for different GCTs and have varying levels. Therefore many CT scans are needed (expensive, radiation stress). Instead can look for circulating miRNA as these are less likely to be degraded than mRNAs (which are v long). GCTs have a low mutation rate (especially testicular), so looking for tumour DNA isn't good.

Question 84

What are the advantages for using miRNAs as a biomarker?

Answer 84

Tumour cells release exosomes into the microenvironment and blood. miRNA levels in the serum are very stable and can be left at RT for a day with no change, and freeze-thawed. In a case study, found that miR-372 levels correlated with the protein marker normally used. Also correlation between miR-372 levels and tumour size has been found. Trials with 4 miRNAs have found high specificity and sensitivity - looks promising! Can track relapses as well

Question 85

How can miRNA be used in diagnosis of brain tumours?

Answer 85

If the tumour is marker negative, would have to do a biopsy to determine if it malignant - this can upset sodium levels. Found that miRNA can be used as a marker and that it is proportional to tumour size. Even when levels have been normalised to tumour size still see significant increase.

Question 86

How can miRNA be used in differentiating between 2 pituitary thickening diseases?

Answer 86

LCH or malignant GCT. If diameter is too small for a biopsy, can look at miRNA levels (if no other markers). Have found that if there are high miRNA levels can keep tracking the tumour and see growth - a neurosurgeon would then go in to biopsy/remove.

Question 87

Why is it important to improve diagnosis of GCT?

Answer 87

Still high cure rates with delayed diagnosis, but has more chance of becoming metastatic and can affect memory and behaviour

Question 88

What are the pitfalls in using miRNA to look for GCT?

Answer 88

Serum volume is dependent on size - if a small tumour in a big person have to look very carefully. May be better to look at spinal fluid

Question 89

How can miRNA be used in diagnosis of other tumours that GCT?

Answer 89

Unclear. Many things can effect the results such as tube used to collect the sample, method of collection, same normalisation, spin speeds (aberrant results at high spin speeds). In GCT studies all of these have been the same

Question 90

When does genomic recombination occur in normal development?

Answer 90

VJD recombination | Meiosis for progeny fitness and evolution

Question 91

What are the types of genomic instability?

Answer 91

``` Chromosomal instability (CIN) - structural (deletions, duplications, translocations) or numerical (aneuploidy/polyploidy) Micro satellite instability - changes in number of repeats, can get double strand breaks Base pair mutations ```

Question 92

What causes genomic instability?

Answer 92

Failure of DNA repair Failure of checkpoints Failure of chromosomal segregation

Question 93

What has Lynch syndrome taught us about genomic instability in cancer?

Answer 93

Is hereditary non-polyposis colon cancer. Found micro satellite instability and tracked this to mutations in mismatch repair genes. Same mutations are found in 15% of sporadic cancers

Question 94

What has HBOC taught us about genomic instability in cancers?

Answer 94

Hereditary breast and ovarian cancer. Found mutation in BRCA1 and BRCA2, involved in DNA damage response and DNA repair (homologous recombination repair)

Question 95

What do BRCA1 and BRCA2 do?

Answer 95

BRCA1 is part of the sensor complex for double strand breaks BRCA2 is part of the repair complex, may be a scaffold protein. If homologous recombination is defective get more non-homologous end joining which is much less stable.

Question 96

How does homologous recombination work to repair double strand breaks?

Answer 96

Break is degraded to get an overhang. Single strand invades the other chromosome and anneals, get DNA replication induced by the break.

Question 97

How do we know genomic instability in cancers isn't caused by rapid cycling?

Answer 97

Different tumours have different patterns of genomic instability - suggests there are specific mutations in certain repair pathways.

Question 98

What mutations are found that cause failure of DNA repair?

Answer 98

In hereditary cancers (Lynch syndrome - MIN; HBOC - BRCA) Very little found in sporadic cancers - mutations not often found in targeted studies. Suggested that a full mutation would be too much instability - there could be more subtle changes on an epigenetic/gene expression level.

Question 99

What mutations are found that cause failure of checkpoints?

Answer 99

p53 deletions as a DNA damage checkpoint - suggests that this could cause genomic instability. However, still get precancerous lesions with genomic instability and normal p53.

Question 100

What is oncogene induced DNA damage?

Answer 100

Suggestion that activated oncogenes and growth pathways could lead to genomic instability through DNA replication stress. Mechanism is unknown.

Question 101

How do failures in chromosome segregation cause genomic instability?

Answer 101

If there are problems organising the spindle (e.g. multiple centrosomes) could get CIN and cancer. In 2010 found that 25% of genome is affected by arm/whole chromosome somatic copy number alterations. If get incorrect segregation get polyploidy/aneuploidy

Question 102

What is euploidy?

Answer 102

Normal number of chromosomes within a cell for a species

Question 103

What is aneuploidy?

Answer 103

Having an abnormal chromosome number

Question 104

What is polyploidy?

Answer 104

Having one or more extra sets of chromosome

Question 105

How can cells gain or lose chromosomes?

Answer 105

Mitotic checkpoint defects - no division of sister chromatids at anaphase Cohesion defects - premature loss of sister chromatid cohesion Merotelic attachments - is increased if microtubule depolymerising kinesis 13 is inactivated (found in CIN) Multipolar mitotic spindles

Question 106

How do multipolar mitotic spindles cause genomic instability?

Answer 106

Can get division into more than 2 cells - cells tend to die by apoptosis as not enough chromosomes to survive Or get clustering of 2 MTOCs at one of the poles and minor chromosome missegregation. Get more lagging chromosomes as there are more merotelic attachments.

Question 107

How do extra centrosomes form?

Answer 107

Centrosome amplification (more than 1 daughter centrosome to each mother) Fragmented pericentriolar matrix can nucleate microtubules and form more poles Loss of centriole cohesion Over expression o fPLK4 leading to increased number of centrosomes at subsequent divisions

Question 108

How can cells become tetraploid?

Answer 108

Cell-cell fusion (viruses) Cytokinesis failure Mitotic slippage (prolonged arrest) Endoreduplication (in megakaryocytic differentiation) If there are 4 centrosomes in a tetraploid cell, get more lagging chromosomes than if only 2.

Question 109

What are micronuclei?

Answer 109

Formed by mis-segregated chromosomes. Either persist as cell divides or are reincorporated into the nuclei. Undergo asynchronous DNA replication resulting in DNA damage and chromosome fragmentation - may explain chromothripis observations in some cancer cells

Question 110

What is endoreduplication?

Answer 110

In which the cellular DNA is replicated but mitosis doesn't occur. Could be due to persistent DNA damage signalling such as telomere damage.

Question 111

How can endoreduplication arise?

Answer 111

Combined loss of p53 and Rb can lead to endoreplication and polyploidy and proliferation

Question 112

How can prolonged mitosis lead to DNA damage?

Answer 112

Depletion of the anti-apoptotic protein MCL1 causes activation of DNase CAD which cleaves decondensed DNA loops. Sister chromatid cohesion is gradually lost which promotes premature separation and merotelic attachment. These processes can lead to chromosome breaks.

Question 113

How can lagging chromatin cause cytokinesis failure and DNA damage?

Answer 113

Can get stuck in bridge and causes abscission failure and bridge stabilisation Can cause regression of the furrow and tetraploidy Can get double strand break and chromatin cleavage

Question 114

How is abscission during cytokinesis prevented in the presence of lagging chromatin?

Answer 114

Aurora B controls the activity of ESCRT-III to delay abscission in the presence of DNA

Question 115

What is the evidence for aneuploidy facilitating tumourigenesis?

Answer 115

SAC-deficient mice with high levels of aneuploidy show an increase in carcinogen induced tumours. Also, over expression of some mitotic components can promote tumourigenesis in these mice.

Question 116

What is the evidence that aneuploidy has no link to tumourigenesis?

Answer 116

Tumours only form in a fraction of aneuploid animals and humans - no direct correlation. SAC-deficient mice have high levels of aneuploidy but no increase in spontaneous tumourigenesis.

Question 117

What is the evidence that aneuploidy suppresses tumourigenesis?

Answer 117

If there are mutations in tumour suppressor genes (p19, Rb, Apc) alongside aneuploidy get suppression of tumourigenesis

Question 118

Does aneuploidy cause tumorigenesis?

Answer 118

Probably no. Thought that it may facilitate tumour development in the presence of other insults though. Can also suppress tumourigenesis in some circumstances - thought to be due to levels of genomic instability being higher than the threshold compatible with cell viability.

Question 119

What is the evidence for polyploidy facilitating tumorigenesis?

Answer 119

in p53 deficient tetraploid mice mammary epithelial cells get CIN (structural and numerical). These cause tumours in nude mice when transplanted in A midbody kelch protein for cytokinesis is associated with Hodgkins lymphoma and binucleate cells In the progression from Barretts oesophagus to adenocarcinoma can do a normal Vogelstein model OR lose p53 then get genome doubling Tetraploid cells display more CIN

Question 120

What is the chromosomal translocation associated with Hodgkins lymphoma?

Answer 120

Reciprocal between chromosome 2 and 3, break point in the middle of the KLHDC8B gene - depletions in this gene have shown to give failure to cytokinesis and binucleate cells

Question 121

How can oesophageal adenocarcinoma form from Barretts oesophagus?

Answer 121

Comparing cells before and after tumourigenesis. Either see a traditional Vogelstein method in which oncogenes accumulate OR a loss of p53 and then gene doubling events.

Question 122

What is the theory as to why polyploidy may facilitate tumourigenesis?

Answer 122

Can cope with more mitotic errors and chromosomal instability. Can also cope better with chemotherapeutic drugs (seen in tetraploid cells in culture) Is associated with poor prognosis in colon cancers

Question 123

What is the evidence for supernumerary centrosomes facilitating carcinogenesis?

Answer 123

Centrosome amplification by over expressing SAK/Pik4 promotes tumourigenesis in drosophila Mutation of genes required for centrosome formation can promote tumorigenesis Centrosome amplification (if with an inducible promoter) can promote tumorigenesis in the presence or absence of p53 Centrosome amplification can induce oncogene like cellular invasion

Question 124

What is the evidence against supernumerary centrosomes facilitating carcinogenesis

Answer 124

2 contradicting papers - one found that over expression of Plk4 didn't cause tumours even with lack of p53 or treatment with carcinogens. Other found that induced Plk4 over expression did promote tumorigenesis with or without p53. Second author used an inducible promoter - potentially suggesting that a bit of extra centrosomes are tumour permissive but too much is detrimental.

Question 125

How was it shown that centrosome amplification by over expression of SAK/PIk4l promotes tumorigenesis in drosophila?

Answer 125

Neuroblasts with extra centrosomes form bipolar spindles during mitosis Higher levels of aneuploidy in SAK over expressed cells Failure to divide asymmetrically in neuroblasts, suggesting that tumour formation could be caused by impaired asymmetric cel division and stem cell over proliferation

Question 126

How was it shown that mutation of genes required for centrosome formation can promote tumorigenesis in drosophila?

Answer 126

SAK or dsas-4 mutations promoted tumorigenesis in brain tissue - not in other somatic tissues though in which cells were dividing symmetrically. Perhaps need impaired asymmetric cell division to see this - get asymmetric cell division in the stem cells of the brain Cells have chromosomal instability (aneuploidy)

Question 127

How can centrosome amplification induce oncogene like cellular invasion?

Answer 127

Activates Rac-1 and disrupts cell-cell adhesion

Question 128

How can CIN be exploited in anticancer therapy?

Answer 128

Is associated with drug resistance Aneuploidy correlates to immune evasion and reduced response to immunotherapy CIN is associated with poor prognosis in some cancers - although extreme CIN can be a good prognosis marker (above a threshold level of genomic instability)? No linear trend - need to understand better first.