Cancer genomics Flashcards
What are the 2 next gene sequencing technologies likely to come into mainstream use?
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.
How does exome sequencing work?
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.
How does Illumina sequencing work?
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.
What are the problems with sequence alignment in Illumina?
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
How can misalignments in Illumina sequencing be verified?
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.
How can cancer mutations be found by non-genome methods?
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
How can cancer mutations be found by genome-wide methods?
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)
What are the advantages and disadvantages of using cytogenetics to look for chromosome translocations?
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
What has CGH arrays taught us about cancer genomics?
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.
What have Sanger sequencing screens taught us about cancer genomics?
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.
What is the significance of TTC28?
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.
How does LINE1 move around the genome?
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.
What is the current knowledge of cancer genetics?
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
What are the different levels of transcription regulation?
DNA state (histone code, DNA methylation, chromatin structure)
Transcription factors
Co factors
Protein post translational modifications
RNA Pol II mediated gene expression (near promoters)
Describe the histone modifications found at enhancers and promoters?
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.
How can enhancers be identified?
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.
What is special about nuclear receptors?
Are the only transcription factors that can be constituently switched on by a ligand.
Describe the structure of nuclear receptors
Have a variable section at the N terminus, then a DNA binding domain consisting of 2 zinc finger domains, then a ligand binding domain.
How can nuclear receptors be drugged?
The ligand binding domain is unique and therefore druggable
How do nuclear receptors act in cancer?
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.
How do cofactors work with nuclear receptors?
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.
How can the binding points for ER be found in an unbiased way?
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.
What did the discovery of where ER binds tell us?
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).
What is FoxA1?
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.
How was it found which ER binding site activated which gene?
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)
What is GRO-seq?
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
How did GRO-seq inform us about ER activity?
27% of whole genome is transcribed when ER is active. Includes enhancer RNAs along with ncRNAs, antisense transcripts, divergent transcripts etc
What do eRNAs do?
Thought to aid chromatin looping - forming bridges between loops. If they are silenced get loss of cell growth and abnormal gene expression.
How is the TMPRSS2-ERG fusion associated with nuclear receptors?
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
How can DNA properties be drugged in cancer?
5-aza-cytodine is a DNA demethylation agent (need to control dosage)
G-quadruplexes may be able to be targeted by inhibitors
How can transcription/pioneer factors be drugged in cancer?
Could regulate other transcription factors as a decoy or block protein-protein interfaces (hard to get into the cell)
How can co-factors and associated proteins that are mutated in cancer be drugged?
AIB1 is a breast co-factor where a drug has been found
IBET inhibit BRD4 co-factors
What factors increase the risk of breast cancer?
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)
What is the normal function for nuclear receptors?
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.
How does ER cause cell growth in breast cancer?
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
How does Tamoxifen affect ER activity?
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.
How does aromatase affect ER activity?
Oestrogen is metabolised from precursors by aromatase. If aromatase is inhibited, it limits the amount of oestrogen that can form (increases hot flushes etc)
How does anti androgens affect AR activity?
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.
What did we learn from mapping AR binding sites?
Loads of AR binding events, like ER, in prostate cancer cells; very few promoters bound.
Forkhead motifs were also enriched
GATA motifs were enriched
How does blocking FoxA1 binding affect ER binding?
No ER binding at all; no gene expression, no cell growth
How does blocking FoxA1 binding affect AR binding?
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)
How is FoxA1 associated with breast cancer outcome?
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.
How does drug resistance arise in ER brest cancer?
Phosphorylation of ER, ER mutations to prevent drug binding (in ligand binding domain). Often found in metastasis.
Why is FoxA1 a good drug target?
Is absolutely necessary for ER+ cancers, even in drug resistant cells. Also needed in AR+ cancers and castrate resistant prostate cancer.
Can we drug FoxA1?
Maybe. Has a restricted tissue expression profile
How do prostate cancers become drug resistant?
Select splicing variants with different ligand binding
What are molecular apocrine breast cancers?
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)
How can molecular apocrine cancers be treated?
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.
How does progesterone interact with the ER pathway?
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).
What is a new way of treating ER+ cancers?
Giving progestins with ER inhibitors such as tamoxifen seems to be a potent way of treating cancer. This is in clinical trails now.
What is the problem with treating cancers with progestins?
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
