14. cancer, iPSC reprogramming and de-differentiation Flashcards
where do tumour initiating cells come from?
uncontrolled proliferation of stem cells/progenitor cells/differentiated cells
over proliferation does not necessarily mean cancer in stem cells but what may it mean?
build up of mutations more rapidly
why are tumour initiating cells less likely to come from differentiated cells?
they are post mitotic and a lot more reprogramming is required in these cells
what can be learnt from reprogramming in relation to formation and proliferation of cancer stem cells?
they give us insight into how cancer can be initiated
what are pluripotent stem cells?
stem cells that are able to give rise to ll three germ layers
what are multipotent stem cells?
stem cells that are tissue specific
what is Waddington’s epigenetic landscape? and what does this landscape imply?
a metaphor for how gene regulation modulates development
>as changes in gene expression and epigenetics occur, the cells has limited options on what it can become (becomes more lineage restricted)
- that cells cannot go back up the landscape
what was their evidence for soon after Waddington’s epigenetic landscape was established?
it possible for cells to de-differentiate and climb back up the mountain
what did John Gurdon do in the 60s?
nuclear transfer
>cloned the first animal by transplanting somatic cells nuclei into a Xenopus oocyte
what was shown about oocyte cytoplasm?
components of the egg cytoplasm are sufficient to reprogram the transplanted nuclei into a pluripotent state
what did John Gurdons experiments also show?
that every cell in the body contains the entire genome
when was the first mammal cloned?
1996 - Dolly
when were iPSC first developed?
2006
what are the four Yamanaka factors
- Oct4
- Klf4
- Sox2
- c-Myc
what can iPSC be used for?
modelling disease and testing therapeutics
what was in vivo reprogramming shown to do? and how was this done?
cause teratomas and iPSC with totipotent features
>transgenic mice that can express the OKSM factors in all cells.
what are teratomas?
these are cancers which form from cells that resemble all three germ layers.
tumours can form in many different tissues in this mice when OSKM factors are expressed in mice. what is interesting about this?
even when there are no immature cells in a tissue tumours can still arise here i.e. reprogramming took place of fully differentiated cells in vivo
what are the key barriers for reprogramming and tumour initiation?
DNA methylation
histone modification
compacted chromatin - lineage restricted TFs they recruit complexes like polycomb and HP1
how is DNA methylation a barrier to reprogramming?
it most stable epigenetic mark
what was though about DNA methylation until recently?
it was lost through cell division
what discovery suggested that DNA methylation could be activity lost?
presence of 5-hydroxymethyl cytosine (5hmC)
what two family of proteins have defined to convert methyl cytosine back to cytosine?
TET -convert 5mC to 5hmC (5-hydroxylmethyl cytosine)
TDG - reconstitutes cytosines (makes abasic site and allow for base excision repair to place)
how are TET proteins activated in the reprogramming process?
by OSKM factors
when TET and TDG are KO, what does this mean?
reprogramming is not possible
what can replace Otc4 in reprogramming?
TET1 - ability to remove 5mC marks is essential for reprogramming
are tet proteins up-regulated in cancer? what might affect this?
you would expect it to be upregulated to removed methylation, but it is not
>people look at cancer once it has established - TET may be present in initiation stage but this is v hard to study
what is mutated in several types of leukaemia? and some cases of acute leukaemia?
TET
DNMT3A (the enzyme which adds methylation)
what is dramatically reduced in human breast, liver, lung, pancreatic and prostate cancers?
5hmC levels
why might tumour cells want low expression of TET?
> cancer cells, once formed, can try to prevent any changes in DNA methylation (inhibit differentiation and maintain division)
so that they can keep their tumour supresses supressed, TET might removed suppression
what can inhibit the function of TET and lysine demethylases?
metabolic changes
what are neomorphic mutations? and where might they occur? and what is this in terms of cancer?
mutation that gives protein/enzyme new function
>IDH lead instead to the accumulation of 2-GH
>oncometabolite inhibits TET and lysine demethylases
- this is another way that cancer cells can limit plasticity and DNA methylation changes in cells
what does this mutation in IDH lead to?
- hypermethylation throughout genome
- increase of stem and progenitor cells relative to differentiated cells
IDH is mutations in 80% of gliomas but TET is rarely mutated why might this be?
the brain might rely more on histone modification to limit plasticity rather than DNA methylation
what ability is often lost once a tumour is initiated?
the ability to removed DNA methylation (e.g. TET activity) - reduces capacity to differentiate and keeps cells dividing in constant state
why might TET activity may inhibit tumour growth? but what does repression of TET and IDH gene allow?
it may allow the cells to differentiate
>it will have the ability to change in its microenvironment
>give tumour advantage to adapt to perturbations in the environment e.g. chemotherapy/moving to different parts of body
what can happen if cells have limited plasticity (in terms of treatment)?
you can use cancer stem cell targeted therapies and kill all the cancer stem cells then the tumour will not have the ability to continue to grow
why does relapse occur in traditional therapies?
some cells (including cancer stem cells) may be left behind
what can happen is cells have plasticity (in terms of treatment)?
if cells in the tumour can regain some plasticity then even when the tumour is targeted with cancer stem cell targeted therapy, the cancer stem cells will die, but some cells can then de-differentiate into new cancer stem cells
>patient will relapse due to residual plasticity
what can help tumours evade immunotherapy treatment?
de-differentiation
what is adoptive cell transfer?
immunotherapy in which T cells with receptors that recognise cancer specific antigens are injected into the patient
how can cancer cells avoid adoptive cell transfer?
> T cells recognise and destroy cancer cells
some cancer cells dedifferentiated, they lose tumour specific antigen and therefore avoiding the T cells
tumour able to regrow after therapy
how efficient is Yamanaka’s initial reprogramming method ?
quite inefficient
how can Yamanaka’s initial reprogramming method be optimised?
KO MBD3 – this binds methylated residues in DNA, part of the NurD repressive complex
>KO leads to less compact chromatin
what does Oct4 interact with?
thrithorax complex component WDR5
>WDR5 recruits thrithorax complex to pluripotency gene promoters and cell cycle genes and promotes gene activation
what else does Oct4 interact with?
a lysine demethylase - removes repressive marks on pluripotency genes
over expression of what can enhance reprogramming?
BRM - ATP-dependent remodelling complex
remodels nucleosomes and opens up chromatin
- over expression of BRM also associated with tumour development in prostate cancer
what is not essential for reprogramming, it accelerates the process?
Myc -potent pro-proliferation oncogene that is highly amplified in many cancers
what does myc do in cancers cells?
> ramps up metabolism and allow cells to grow faster
>this generates metabolic intermediates that are essential for reprogramming
does myc function as a hetero or homo dimer?
heterodimer - when myc dimerises with Max this activates gene expression
what happens when Max dimerises with Max and Mad?
this represses gene expression
some cells cannot proliferate if there is no Myc function, what is this called?
oncogene addiction
they made transgenic mouse that can conditionally express the dominant negative form of Myc induced by doxycycline – temporarily inhibited Myc function in mice, what was shown in these mice?
these Myc form homodimers in cells - no Myc left to bind Max to turn on genes to allow cells to divide
what can transient expression of dominant negative c-Myc do?
can stop K-ras induce lung tumours, and lead to their regression
what is a side effect of expression dominant negative c-Myc?
this stops all stem cells from dividing - mice can tolerate this and recover
what therapeutic idea lead from this transgenic mice study?
drug that inhibits Myc from binding Max could mimic this and potentially be revolutionary
is maintaining differentiated state an active process?
recent work has revealed that maintaining a differentiated state can be an active process
give an example of how maintaining differentiation is an active process, and what are these factors often associated with?
mutations of certain key factors can cause cells to de-differentiate into less differentiated state or even stem like state
>tumour formation
give an example of two lineage specific guardians and how perturbing them can result in de-differentiation
> lola ablation causes de-differentiation in immune neurones into NSCs
prox1 ablation causes trans-differentiation of lymphatic cells to blood cells
is differentiation more like climbing up a mountain than rolling down a valley?
as cells differentiate do we need to have barriers to stop them rolling back down?
it may be a bit of both - these factors involved in active maintenance are implicated in how caners initiate
plasticity in gut epithelial cells can lead to tumour initiation, how does this occur?
increased NF-κB and Wnt signalling in post-mitotic intestinal cells causes dedifferentiation
>this leads to hyperplasia
why do people that have inflamed guts get cancer more easily?
they have high levels of NF-κB
recent work has highlighted strong parallels between what two processes?
iPS reprogramming and tumour initiation
what is the ability to alter DNA methylation states closely linked to?
cell plasticity
how is cell plasticity a blessing and a curse to cancer cells?
want plasticity to initiate and then once cancer has formed you want to prevent plasticity - prevent differentiation and keep proliferating
what do different cell types have in terms of intrinsic plasticity and guard against de-differentiation?
different states of intrinsic plasticity and different methods to guard against de-differentiation
