Cancer

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  • Created by: Sarah
  • Created on: 24-05-19 07:23
what is cancer?
a collection of related diseases when cells start to divide uncontrollably and spread into surrounding tissues
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what part of cancer causes death?
1) directly: failure of organs in which it spreads because it takes all the nutrients and energy, controls organ failure such a mass that the organ can't function2) indifrectly: cachexia- wasting away other tissues which then fail
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hallmarks of cancer? (8)
resting cell death, evading growth supressors, enabling replicative immortality , sustaining proliferative signalling genome instability and mutations, activating invasion+metastasis, avoiding immune destruction, inducing angiogenesis
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what 4 things are looked at in histology of examing nuclear architecture to diagnose cancer?
1) high nuclear/cytoplasmic ratio 2) hyperchromatic chromatin (more chromatin-more nucleus staining) 3) nuclear outline 4) size of nucleus
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what are these suggestive of?
major chromatin alterations and an epigenetic component
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what was discovered soon after the discovery of DNA methylation?
changes in DNA methylation in cancer
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how do they discover the change in DNA methylation in cancer?
Hpa II hypersensitivity- did a restriction digest, gel electophoresis and blotted to see if HpaII could cut because it doesn't cut methylated DNA so fragments would be longer
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describe the Hpa II sensitivity method they used?
got total genomic DNA, restricted with Hpa II or Msp I from either normal or cancer tissues cells then restricted with restriction enzymes, stained with ethidium bromide run a gel and visualise the DNA
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what did they find out about DNA methylation in cancer with phosphate?
lighter products in cancer cells- it had been cut up more as Hpa II could access it, DNA cancer cells not mehtylated -> REs cut into small fragments -> run further on gel -> less methylation in cancer cells
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what does it mean that a hallmark of cancer is genome instability?
there's a lot of rearrangements in the genomes that can detected in these cells
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what does it mean that they're able to induce angiogesis?
make a good blood supply for the tumour both from attracting angiogensis from the nomrla blood system and by making their capillaries but needs a lot of energy
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what is it called that there's less methylation in cancer?
genome wide demethylation/hypomethylation in cancer
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what happened when they compared specific genes in normal and cancer cells?
did a southern blot with antibodies against DNA methylation put the gene on a bead and immunoprecipitate
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what did they see when they looked at methylation of specific genes?
some genes had hypermethylated CpG islands in the 5' regions of some genes which are cancer related
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what was found with methylation overall in cancer cells?
smaller fragments of DNA in cancer cells then nrmal cells- RE could cut- hypomethylation in DNA
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what did they dind out when they looked at probes for 3 different genes in carcinomas?
number of fragments hypomethylates in cancers was higher and even further higher when theres metastasis
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what are the 2 types of changes seen in most cancer?
1) global genomic hypomethylation of most genes (4-60% cpg islands methylated instead of 80%) 2) specific hypermethylation of cpg islands at the 5' end of genes related to cancer-
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what is it called when the CpG islands are hypermethylated in some cancer related genes? what can this be used for?
CpG methylated phenotype- can be used in clinics for diagnosis purposes
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overall change in cancer?
bidirectional changes- most of genome is hypomethylated but some genes are hypermethylated
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what is an example of a gene that is hypermethylated in many cancer types?
CDKN2A
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what does CDKN2A encode?
2 products that result from alternative splicing- 1) P16-INK4A 2) P14 RAF
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What is P16-INK4A?
cyclin dependent kinase inhibitor
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what does the P16-INK4A cyclin dependent kinase inhibitor do?
keeps retinpblastoma in its active state
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what does the inactivation of P16-INK4A do?
leads to uncontrolled cell cycle progression so hallmark of cancer- = evades growth so cells divide uncontrollably
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what is P14 RAF?
protein that initiates P53-dependent apoptosis -
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what is P14 RAF linked to?
resisting cell death + dividing uncontrollably
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what did the first experimental evidence of CDKN2A hypermethylation come from?
bisulfite sequencing of DNA from different samples of normal human oral keratinocytes and diff samples of squamous cell carcinoma cells
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what did they see with the methylation difference of CDKN2A?
normal cells- no methylation, hypermethyation in cancer cells- no sites not methylated
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what does DNA promoter methylation do to gene expression?
inhibits gene expression so it means that in cancer cells these genes will not be expressed
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what is P16A-INK4A usually doing?
cyclin dependent kinase inhibitor keeps rb active, inactivation leads to uncontrolled growth as P16 inhibits CDK4 and cyclin D which inhibit rb and cause cell cycle arrest
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what is P16?
a tumour supressor gene
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what happens if P16 is hypermethylated?
usually keeps Rb active (prevent cell growth) so hypermethylation causes Rb to become inactive and leads to uncontrolled cell cycle progression
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function of retinoblastoma?
prevent cell growth- is a tumour supressor protein
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what does inactivation of P16 lead to then?
evading growth supression
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what does P16 usually do to keep rb active?
inhibits CDK4 and cyclin d which activates rb and causes cell cycle arrest (no uncontrolled proliferation)
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what happens when there's no P16?
no inhibition of cyclin D and CDK 4 so rb gets inactivated and there's no cell cycle arrest so evade cell cycle growth
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what does the other product of CDKN2A, P14 do?
P14 inhibits the interaction between HDM2 and P53 which promotes apoptosis
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what happens when CDKN2A is hypermethylated the bit with P14 inactivaiton?
no P14 -> HDM2 is inhibiting P52 therefore there is no apoptosis - so evade cell death
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what does P14 inactivation cause?
evade cell death
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what other gene is hypermethylated?
BRCA1
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where does the name BRCA come from? what was the first evidence of?
breast cancer- BRCA1+2 were the first evidence that there's a genetic component in the probability of getting breast cancer
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what was done to show there was a genetic component in cancer? what was this before?
before DNA structure was discovered- looked at different female patients with breast cancer and built genetic inheritance trees and they've seen a strong genetic link in familial history of breast cancer
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because they didn't know the DNA structure what were they hunting for
the hunt for the gene causing breast cancer- took 46 years until it was linked to a specific gene with a zinc finger domain which binds to nucleic acids
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does BRCA1 act on multiple pathways
yes- it's at the centre of multiple regulatory pathways
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what are the regulatory pathways BRCA1 acts on involved in?
required for chromatin remodelling, checkpoint regulation, DNA damage,
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what is one of the most important pathways for cancer?
DNA damage response
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why is BRCA1 acting on the DNA damage response important for cancer?
when BRCA1 is hypermethylated it's not functional so the normal DNA damage response pathway is not activated
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what does it mean if BRCA1s hypermethylated then theres no DNA dmaage response pathway activated?
if mutations or rearrangements occur cells can't repair the DNA which is an additional source of mutations- links BRCA1 to another hallmarks of cancer = genome instability and accumulation of new mutations
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what does BRCA1 hypermethylation do to chromatin remodelling?
if BRCA1 is hypermethylated then its inactive normal chromatin remodelling is not happening which causes changes in the epigenetic code which links methylation with histone acetylation
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what does BRAA1 hypermethylation meaning theres not normal chromatin remodelling do?
histone acetylation link
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final gene hypermethylated in cancers?
CDH1
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3 genes hypermethylated in cancers?
1) CDKN2A, 2) BRCA1 3) CDH1
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where is CDH1 normally hypermethylated?
in nearly all carcinomas in the CDH1 gene
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what does the CDH1 gene encode?
E-cadherin
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what does E-cadherin do?
key protein which regulates cell-cell adhesion, one E-cadherin binds E-cadherin on the adjacent cell therefore provides physical attachments between cells
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what does E-cadherin bind to in the cells?
actin cytoskeleton through cadherins
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what does loss of E-cadherin in humans link to?
lots of types of malignant cancers
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why is loss of E-cadherin linked to malignant cancers?
it detaches the primary tumour contacts to neighbouring cells, the cells escape the tumour, enter bloodstream and invade other organs
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what is needed for cells to migrate from the tumour into the blodstream?
detach and lose cell-cell adhesions
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how did they show CDH1 is hypermethylated?
E-cadherin positive carcinoma lines digestions there are no products visible meaning that it cut in so many places it didn't recognise anything anymore so can cut when E-cadherins there,in E-cadherin -ve line products visible RE cant cut hypermethyla
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why is there no cutting and long fragments in the E-cadherin negative line?
because CD1/E-cadherin promoter is hypermethylated so REs can't cut it
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what hallmark of cancer is E-cadherin linked to?
activating invasion and metastasis
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what do these 3 genes hypermethylation show?
hypermethlation of single individual genes and low number of these genes can be directly linked to hallmarks such as evading apoptosis and uncontrolled proliferation (CDKN2A), genomic instability (BRCA1) and invasion and metastasis (CDH1- E-cadherin)
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why doesn't the number of genes have to be large to induce all these changes and hallmarks?
1 gene big impacts eg BRCA1 acts on multiple regulatory pathways- Cpg island methylated phenotype low num of hypermethylated genes as linked to various cancer hallmarks and progression
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what happens to the genome globally?
its hypomethylated
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what causes the global hypmethylation in cancer?
transposable elements
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how much of the human genome consists of transposable elements?
50% of human genome comprises transposable elements
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what do transposable elements do?
are elements that can excise themselves from the genome and move to another place
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how are transposable elments different?
several types of transposable elements- diff in their mechanisms of transitions (how they move within genome)
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what are the mechanisms transposable elements use to move?
some: excising the whole DNA fragment and inserting them into a different place. Others moved by expressing RNA, making DNA from it and inserting the DNA into another place
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what do transposable elements differ by?
their organisation and their distribution in the gneome
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what is one class of transposable element particularly important in cancers?
LINE elements
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what are LINE elements?
long interspersed nuclear elements
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what did most transposons in the human genome lose? what kept this?
LINE elements retained their ability to be active mobile elements but many transposable elements lost this
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what do LINE elements encode?
genes required for transposition
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how much of the human genome do one class of LINE elements (LINE 1 sequence) make up?
17% of the whole human genome
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what happens to LINE elements in normal cells?
LINE elements are normally highly methylated so they cant express enzymes which are recquired for their movement- so they stay in the same place
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what happens when there's hypomethylation of LINE elements in cancer?
they can transcribe the enzymes needed for transposition- many of them located in a normal gene so can be transcribed and produce short RNA sequences
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what are the short RNA sequences they make complementary to? why?
normal mRNA- will act as microRNAs which cause post transcriptinal degradation and mRNA decay so normal proteins not expressed
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what other thing can they produce? why?
transposases- important for their transposition within the genome- start moving the element
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what does it cause when a LINE element is excised?
a double stranded break
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why dwon't this double stranded break be fixed?
other genes like BRCA1 inactivated- no normal DNA damage response pathways
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what happens to the double stranded break?
it can't be repaired and causes deletions, truncations and genome instability
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what will genome instability?
induce new mutations in the genome and promote cancer
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what happnes if they move into a gene where there was no transposable element before?
will induce mutation and alter expression of that gene
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what is causing global hypomethylation and hypermethylation of specific CpG islands?
not fully undertood but IDH2 and IDH1 have been linked to changes in DNA methylation
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what do IDH1/2 do?
they modulate the products which are required to put methylation marks on DNA
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what happens when there's a mutation in IDH1/2?
they balance of metabolites required for DNA methylation are altered so alter efficiency of methylation and demethylation enzymes and results in a whole genome change in DNA pattern which contributes to tumourigenesis
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when do DNA methylation patterns appear in cancer?
very early in cancer
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what is the overall view of cancer?
a tone point a mutation happened which promotes the growth of cells and then additional mutations are acquired over time provide advantage and induce invasion- accumulation of mutaions
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what is their feedback between?
mutations, DNA methylation mutually altering each other which leads to cancer metastasis and cell and patient death
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changes in DNA methylation pattern are often due to what?
mutations in DNA methylation writers or erases
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why is there a problem in studying histone modifications in cancer?
different types of cancers - have different histone mods, its not a single histone mod but its all different ones in diff groups in diff cancer types
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why is it narrow minded to look at the histone lysine 9 acrylsation in the lung histone modification?
this is only what the researchers looked at not all that's changed in that cancer
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what did the table of histone mods and diff cancer types show?
multiple histone modifcations are altered in multiple cancers
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what don't we know?
we don't know all of the histone modifications, news mods being discovered every year
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when researchers looked a a wide range of modifications and samples for each of the modifications what did they find?
different patient had different alterations in histone mods- some had high levels, some low and there was some correlation with the severity of the disease and prognosis
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what was suggested?
that higher levels or stronger alteration of the histone modification profile was associated with high severity of cancer- but it wasn't clear cut
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how are polycomb complexes quite simple in drosophila?
lower number of genes- have PRC1 + PRC2 and many of the genes are represented by a single copy
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what happened during the evolution of vertebrates and humans?
2 events of genome duplication so many genes in flies have 1 but have 3 or 4 in vertebrates
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what is the PRC2 complex like in vertebrates?
more vaired- has at its core 2 lysine methytransferases (Ez homologue 1+2)
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what are enhancer of zest homologues 1 + 2?
mutually exclusive- so PRC2 either has one or the other not both
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how are Ez homologues 1 + 2 very similar?
they methylate H3K27 and require stable asosciation with Eed and Suz12 (extra sex combs paralogue and supressor of zest paraogues)
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so PCR2 has a distinct core with either Ezhz 1 or 2 but what else do they have?
form distanct complexes with distinct biochemical functions by recruiting additional redudant subunits- this requirement is occurring in a cell type dependent manner
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they have a core and each core can have an additional what? how many possible option does this make?
2 core options (Ezh1 or 2), and can have an additional RBP4 or RBP7 = 4 options
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what else does it finally recruit?
proteins such as JAIRD2, PCL1/2 or 3 which are mutually exculusive and AEBP2
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how is PCR2 more complex than a fly PCR but more simple than PCR1?
some variability in the composition- 2 cores some alternative components but not that many
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what has much more nbiological complexity?
PRC1 in vertebrates including humans
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what does the PCR1 core consit of?
RING1A or RING1B = they are mutually exclusive
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what does the PCR1 core require stable association with?
one of the polycomb group ring fingers = PCGF proteins
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what can the classification be based on?
which PCGF (polycomb group ring finger is involved)
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how many polycomb group ring fingers are there?
6
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what do PGCF 2 + 4 further recuit?
either cbx or YBP protein which makes these 2 subgroups = mutually exclusive again
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how many different forms of PRC1 is there thought to be?
at least 100 different forms- that could functionally exist in a given cell at a given developmental stage
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why are polycomb complexes so hard to study in humans?
over 100 different complexes could exist and many of them potentially having redudant functions and being able to compensate for each other so mutating one protein wouldn't be helpful
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where is most of our information abotu PRC1 coming from?
fly studies where the complex is much less complex
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PCR2 in vertebrates similar to flies is essential for what
development
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what did the KO of Enhancer of zest homologue 2 in mice show?
at that developmental stage becomes a mass of cells without any clear structures, no differentiation and they generally die very soon after implantation = no proper development
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what has PCR2 been show to be really crucial for in a number of studies?
regulating pluirpotency, differentation
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what does KO of PRC2 lead to?
spontaenous increase in developmental gene expression, differentation defects and premature lineage specification
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why does the premature lineage specification not occur correctly?
its not accompanied by efficient silencing of pluripotency genes and its not properly specficied
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what is PRC2 really important for balancing?
PCR2 is really important for balancing proliferation and differentation
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what happens to pluripotency in normal development?
pluripotency is lost and differentation is induced
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what happens to pluripotency in the EZHZ KO?
pluripotency is not really lost or completely turned off, differentation is prematurely started which leads to a total disbalance in expression and cell cell behaviour
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what does PRC2 overexpression correlate with in several types of cancer?
PCR2 expressin correlates with poor cancer prognosis
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whats the expression of EZ homologue like in normal tissue?
very low expression of EZHZ in fully differentiated cells
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what is the expression of EZHZ like in th breast carcinomas?
EZHZ is severely overexpressed in breast carcinomas
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how was it shown to be overexpressed in breast carcinomas?
WB- no EZH2 protein observed in the nromal tissue, in invasive BC samples EZH2 clearly detactable and in metastatic prostate cancer a lot of EZHZ2
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what is overexpression of EZHZ 2 linked to?
poor prognosis and more metastasisng the cancer is- cancer is worse
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what did the microarray samples from cancer samples show?
metastatic cancer is much redder- more expression of EZHZ2 in metastatic cancer then localised cancer comparative to benign or normal tissue
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if the overexpression of PRC2 leads to a poor prognosis what does PRC2 inactivation do
also leads to poor prognosis
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how was it shown that you get poor prognosis with PRC2 inactivation?
myeoproliferative neoplasm had a mutated PRC2 then the general prognosis of patients is much worse than if PRC2 is unmutated. More patients alive after months of diagnosis worse when PRC2 mutated (inactivated)
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what types of mutations were observed in these patients?
copy number alterations or unipaternal disomy
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is too much or too little PRC2 bad?
yes
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what mutations in PRC2 component genes are widespread in cancer?
both GOF and LOF
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why are different types of cancer classified differently depending on the cell type they originated from?
even if the cancer originated from the same tissue they can be produced from different cell types which can affect the cancer behaviour which is why they're split into further subtypes
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how was it shown that both GOF + LOF alterations in genes encoding PRC2 compoents are widespread in cancer?
combined data from multiple cancers, all diff alteration and frequency of alterations in the PRC2 gene eg amplification of the gene 2 or more normal copies is overexpressed (more mRNA, more protein), splicing and missense mutations, looked at G/LOF
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what could missense and splicing mutations do to PRC2?
alter the function of this protein by increasing or decreasing activity, some splicing mutations cause mRNA decay and no protein
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what did they find?
across different cancer types and subtypes both GOF and LOF of PRC2 is observed
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what kind of role does PRC2 have?
an oncogenic role
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how can PCR2 overepression affect P16?
too much PRC2 -> hypermethylated P16 -> stops P16 being expressed P16 won't inhibit cyclins -> Rb inactive -> cell cycle progression evade aptosis uncontrolled growth
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What else does PRC2 inhibt?
E-cadherin which connexts cells so lose contact with primary tumour and form metasis
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what did immunofluroscence of E-cadherin and EZHZ2 in human breast tumour tissueshow?
cells mutually either have high EZH2 in the nuclei or E-cadherin. EZHZ2 expression is mutually exclusive with E-cadherin- 1 or other
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what is it likely that this mutual exclusion expression fo EZHZ2 is doing?
likely that one inhinits the other
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hwo did they prove that EZH2 inhibits E-cadherin expression?
mutated and modified EZHZ2 or directly overexpressed EZHZ2. Showed that EZHZ2 was directly inbhibiting E-cadherin expression
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what is notch signallign important in?
very important in development but also cancer progression
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what does notch receptor bind to?
different ligands such as delta/jagged. Ligand and receptor are TM so need cell-cell contacts to have notch signalling
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what happens upon signalling (notch binds delta)?
activity in the notch signalling domain which leads to release of notch IC domain that goes to the nucleus and acts as a TF to activate or repress target gene expression
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what is one of the cancer types really linked to alterations in notch cell?
T acute lymphoblastic leukaemia- hematologic maligancy (WBC cancer characterised by activating mutations in NOTCH1 gene)
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what happens with the activating mutations in NOTCH1?
notch doesn't need a ligand it activates the pathway anyway- signalling is activated all the time
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where does notch predominantly bind?
unmethylated histone 3 lysine 27 - doesn't tell us whether its just binding H3K27 or whether its binding and helping to remove this mod
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what was shown in hyperactivated notch tumour cells?
EZHZ2 on the DNA is strongly reduced (compared to normal cells) so where notch is overactive you get loss EZHZ2 on the DNA
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what did this all lead to the model of?
when notch signalling pathway is consistutiely activated due to a mutation, notch binding mediates loss of histone 3 lysine 27 methylation
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how does notch CA signalling lose Histone H3 lyisne 27 methylation?
eviction of PRC2 so it can't make these marks
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what is the consequence of loss of H3K27me and eviction of PRC2 by notch1 signalling?
expression and activity of notch signalling is even further elevated/activated
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what is there a feedback loop between?
notch enhancing its activity and helping promote its own function by eviction of PRC2
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what happens if PRC2 is overexpressed?
notch eviction of PRC2 will be less efficient, it cannot be evicted by notch that efficiently and H3K27m methylation wouldn't be that affected by notch signalling, notch sig cant promote itself
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in this case what is the overexpression of PRC2 doing in NOTCH activated cancers?
overexpression of PRC2 is actually preventing the development of cancer
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what does this tell us
depending on the context and underlying mechansims that form the cancer diff things will help usually PRC2 overexpression bad but when cancer caused by overactive notch slows cancer ptogression
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why is PRC2 a good target for cancer treatment?
PRC2 mutations are really frequent in cancer and because its an enzyme its easy to find inhibitors for these enzymes
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what was found with mutant lymphoma cells?
inhibition of EZHZ2 blocks H3K27 methylation and actually kills mutant lymphoma cells- stops P16 and E-cadherin being overmethylated so prevents cancer
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when would inhibiting EZHZ2 help?
only in cases where too much EZHZ2 promote cancer- wouldn't help where NOTCH is hyperactivated as in this cancer inhibiting PRC2 makes the prognsois worse
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what do we need to treat cancer?
personalising medicines- complex heterogenous diseases like cancer so need to undertsand the exact mutations in patients to get the best treatment
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what clinical trials are happening in cancer?
phase 2 clinical trials- they test EZHZ inhibitor tazemetostat for particular subsets where the cause is known sarcomas and b cell lymphomas
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what can understanding the genetic causes in individual cancers hopefully lead to?
inhibitors to give specific treatments
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