17. metastasis Flashcards
name the process by which tumour cells exit the blood stream?
extravastation
describe the basic structure of intestinal tissue
- epithelial cells absorb things from the gut
- there is muscle and connective tissue under this
- these cells are embedded within capillaries and blood supply
- under this is a layer of epithelial cells which sit on the basement membrane and proliferate out from this
- it is organised and regular
where is a tumour most likely to arise in the gut?
in the epithelial layer
made the two types of adhesion molecule that cells use
cadherins and intergrins
what type of adhesion molecules are used for cell to cell adhesions?
cadherins
what type of adhesion molecules are used to cell to ECM adhesions?
intergrins
what type of signalling in the soft agar assay has been disrupted which means normal cells can no longer proliferate?
intergrin
what type of signalling in the focus formation assay has been disrupted which allows cells to grow regardless of contact with other cells?
cadherin
cells that are adhered to a basement membrane, for example tissue culture plastic, have intergrin signalling. what expression is induced in these cells? and how does this change when cells are in suspension?
cyclin A
cells do not express cyclin A when in suspension
expression of what is elevated when cells are in suspension and there is no intergrin signalling? and what happens to this when cells are reattached to a membrane?
p27
levels rapidly decrease once cell is attached to the membrane
there is a link between intergrin signalling and what?
cell cycle machinery
what are the relative sizes of the intra- and extra-cellular portions of intergrins and cadherins?
large extra-cellular domain and a small intra-cellular domain
when intergrins interact with ECM, what happens in the cytosol of cells?
intergrins interact with actin filaments and signalling molecules
intergrins are heterodimeric proteins, describe the possible subunits and comment on the combinations
- there are 15 alpha subunits and 8 beta subunits
- different combinations of these dictate the proteins that they bind on the outside of the cell, and this binding dictates whether or not there will be intra-cellular signalling
name a molecule that is a key player in intergrin signalling and what outcomes does it modulate? (3)
ILK - intergrin like kinase
- it modulates cyclin D levels and proliferation
- it can modulate the pro-proliferation, pro-survival signal ALK
- cell motility
what happens if ILK is over expressed?
it becomes oncogenic, the more ILK the more malignant the tumour
when you deregulate ILK, you compromise you ability to withstand cancer
what is responsible for cells being able to recognise their neighbours and why is this useful?
cadherins
if they can sense their neighbours then they know not to proliferate into that cell
what do cadherins need in order to bind other cadherins?
calcium
cadherins are calcium sensing
mammary gland tumour cells were introduced into mice in a non-breast location. these were either p53 KO or p53 and cadherin KO, what was observed in each case? what does this show?
p53 KO cells are not sufficient enough to cause tumours
p53 and cadherin KO cells proliferate and metastasise and spread throughout the body and are selective in where they go
this shows that KO cadherin allows expansion and metastasis
how can injected tumour cells be traced in a mouse?
loads them with an enzyme that can create bioluminescence when given the appropriate substrate
there are links between cadherins and actin. what does actin control?
cell shape and motility
cadherin signalling can results in the formation of three things, what are they?
- stress fibres
- lamellipodia
- filopodia
how does the cadherin structure cause changes in the actin structure?
by regulating a family of small GTPases
when the cadherin complex inhibits Rho what does this lead to?
stress fibres form, preventing cells from being motile
when the cadherin complex activates Ras what does this lead to?
Lamellipodia check surroundings, if everything is ok and there is a monolayer of cells then cells do not proliferate
if not then they will proliferate to rectify it
when the cadherin signalling activates cdc42 what does this lead to?
Filopodia are more elongated than Lamellipodia but have the same function to check the environment and check that everything is ok
name three molecules that interact with cadherins and mediate its contact with actin filaments and which more directly contacts the actin filament and what does this mean?
p120 catenin
beta catenin
alpha catenin - alpha catenin directly contacts the actin filament, it can potentially make changes in the cytoskeleton that are independent of beta catenin and p120
which of the three molecules that interacts with cadherins are most directly associated with cancer?
beta catenin
what does beta catenin interact with?
it interacts with cadherins and alpha catenin
what is the homologue of beta catenin in Drosophila called? and describe the structure of this protein
armadillo
it has multiple repeats of alpha helices with turns throughout the protein, these are called armadillo repeats
what makes beta catenin good for binding other proteins?
- large surface area
what do the alpha helices and loop do in beta catenin?
alpha helices hold structure in place while the loops provide binding specificity
where is beta catenin found in normal cells?
at the membrane associated with active cadherins or in the cytosol being recruited to the membrane or degraded
where is beta catenin additionally found in cancer cells? and what type of cancer is this particularly important for?
in the nucleus
this is particularly important for the development of tumourogenesis in colon cancer
what does FAP stand for?
Familial Adenomatous Polyposis
what two forms can colon cancer come in?
familial and sporadic
what happens in FAP?
lots of benign polyps form in the epithelium of the large intestine, these transform into malignant colon cancer if untreated
what mutation is seen in 80% of colon cancers?
APC - anaphase promoting complex which is an important tumour suppresser
how many hits does a TS require to knock it out?
2
how do polyp in the colon arise?
enhanced growth of epithelial layer
name two proteins that APC interacts with
GSK3 and beta catenin
where do most mutations that arise in APC occur and what affect does this have? and what does this imply?
mutations normally occur in the first 1500 codons and this results in APC no longer being able to bind beta catenin
this implies that interaction is crucial for developing colon cancer
what mutation in beta catenin stabilises it? without this what happens?
serine 37 to a residue that cannot be phosphorylated
WT beta catenin has a very short half life
what two proteins bind beta catenin in order to kept its cellular levels low?
APC and GSK3
describe the degradation process for beta catenin
- APC bind GSK3
- APC binds beta catenin
- GSK3 phosphorylates beta catenin at multiple sites (predominantly at serine 37)
- this phosphorylation allows recruitment of Ub-ligase machinery
- poly-Ub beta catenin tagged for proteosomal degredation
what happens to beta catenin in cell of people suffering from FAP?
they have mutated APC and so GSK3 is not brought into close proximity with beta catenin and this stabilises beta catenin, leading to enhanced levels of beta catenin in the cytoplasm
what normally happens to beta catenin in the cytoplasm?
it is either recruited to the membrane or degraded
what type of signal does beta catenin have encoded in it?
a nuclear localisation signal
what does beta catenin interact with once in the nucleus? and what it this?
TCF/LEF
this is a transcription factor that lacks an activation domain
what genes does TCF/LEF target?
cyclin D - cell cycle entry
c-myc - when over expressed leads to tumourigenesis
MMPs - matrix metaloproteases
a criptic transactivation domain is seen in what protein and what can it recruit?
beta catenin
it can recruit basal transcription machinary
why does this beta catenin localisation process occur in normal biology and how it this different from when it occurs in tumourigenesis?
it occurs when there is not enough cadherin forming complexes and so the cell should proliferate
it is normally very tightly controlled
what can inhibit GSK3? and so what happens when you have lots of this?
ILK
when ILK is over expressed, beta catenin is stabilised which leads to nuclear localisation of beta catenin
what three types of mutations can affect beta catenin signalling and are seen in cancer?
- inactivating mutations in intra-cellular cadherin - this results in overwhelming degradation machinery
- inactivating mutation in APC - less beta catenin degraded
- domain activating mutations in Beta catenin (this might be in serine 37)
what type of cadherins are present on early stage tumours? and how it this different to metastatic tumours?
E-cadherins
metastatic tumours often have other cadherins
what does E-cadherin expression in tumours correlate to? and what is this due to?
lack of metastastic behaviour
lots of E cadherin means little mobility
what process to tumour that become metastatic undergo? and what is this?
Epithelial-Mesenchyme Transition
this is a change in cadherin gene expression
for a tumour cell to be able to proliferate, what does it need to deregulate?
its intergrin and cadherin signalling
they need to be able to ignore their surroundings and ignore whether they are on a basement membrane or not
the ECM is a defined structure, what do cells need to produce in order to get through this and metastasise?
Matrix metalloproteases
MMPs have a signal peptide, what does this mean?
this means that when they go through the rough ER they are put into secretory vesicles and secreted form the cell
most MMPs are secreted, what can they also be?
transmembrane proteins, although their function is on the outside of the cell
how many MMPs are found in humans?
around 30
compare and contrast the different MMPs
- they all that the same catalytic fold and each have different accessory domains
- they have different substrate specificity to get through different components of the ECM (e.g. collagen, gelatine and elastin)
describe the catalytic domain of MMPs
they have three histadines that coordinate a zinc ion
there is an additional acid which also contributes to the active site
there are two conserved sequences in MMPs, what are they?
the catalytic site and the control switch which keeps the enzyme in an inactive form
how does the MMP control switch work?
the control switch forms interactions with Zinc through a cysteine residue, this means the that the active site of MMPs is blocked and so they cannot function
where is the control switch and what has to occur in order for MMPs to become active?
the control switch is in the pro-domain, this needs to be cleaved for MMPs to become active
what two things does degrading ECM allow?
it makes a pathway through the ECM and provides amino acids and other factors which the tumour can utilise
what type of amino acids do MMPs cleave? and what is beneficial about this?
fairly hydrophobic amino acids like valine, leucine and isoleucine
they are quire common amino acids and so ECM can be cut into small pieces - i.e. pieces that are not structurally hindering for migrating cells
why are MMPs a potential drug target? and why might this fail?
- blocking their function would reduce cell mobility and metastasis
- there are lots of other proteases that do similar things, blocking MMPs may result in cancer cells using other proteases instead
what does a change in cadherin expression allow cells to do?
find distant location by promoting extravasation and colonisation in secondary site
do tumour metastasise randomly throughout the body?
no, different types of tumours preferentially metastasise to certain locations in the body
how was the seed and the soil theory suggested and what is it?
- it was proposed by a surgeon that did lots of autopsies
- he found specific cancers metastasise to specific locations
- the seeds are the cells and they sew themselves in distant locations, soils that are appropriate for seed to germinate
when a patient suffers from Melanoma, how many deaths are due to secondary tumour formation in the lungs?
40%
what is interesting about where cells metastasise?
it is not dependent on blood supply
give two examples of things that can affect where a cell metastasises?
- their cadherin expression profile
- tumour released factors
what type of cadherin expression promotes liver metastasis? and what is this associated with?
p-cadherin
this is associated with poor prognosis in colon cancer
what two factors can tumour release that affect metastasis?
IL-11 and Lysyl Oxidase
where does breast cancer preferentially metastasise?
to the bone
breast cancer cells that are prone to metastasise express what in high levels?
MMPs and IL-11
what are osteoclasts? and what affect does IL-11 have on them?
- large multinucleated bone cells which absorb bone tissue
- IL-11 recruits and activates them to break down bone so that there is more space for metastatic cells to colonise
why do breast cancers preferentially metastasise to the bone?
breast cancer cells express CXCR4, a receptor for the chemokine SDF1 which is produced by osteoblasts at high levels in the BM
what are the microvesicles produced by cancer cells called and what is their function?
exosomes enter circulation and target specific tissues, they generate a pro-inflammatory environment which is favourable for tumour formation
what is often seen in people that die from breast cancer? and why it this?
- lots more micro-metastasis (single cells) in the bone marrow than was initially thought
- single cells can lay dormant in tissues for a long time
what does a large number or circulating tumour cells suggest? and what is worse than this?
- a worse prognosis
- circulating clusters
what does Lysyl Oxidase do?
- it causes alteration in lysine side chains of collagen which results in the formation of aldehyde cross links
- this is disrupts collagen homeostasis and is favourable for its break down
what does the break down of collagen favour over what? and what does this mean?
- it favour osteoclasts at the expensive of osteoblasts
- this leads to the breaking down of bone which gives more potential to collanise these locations
what are stromal cells? giving an examples
connective tissue cells found in many organs which support the organ
- fibroblasts are one of the most common stromal cells
how are stromal cells implicated in metastasis?
tumour cells from certain locations have been seen to colonise particular stromal cells, the interactions between these cells may affect metastasis
what do different exosomes from different tumour cells target? give an example
different exosomes from different tumour cells target different tissues
exosomes from breast cancer prepare tissues like lung and liver for metastasis
what do exosomes form?
a pre-metastatic niches
what proportion of cells that undergo metastasis die? and what does this tell us about the metastatic process? and what sorts of tumour does this not matter for?
99.98%
this tells us it is a very inefficient process
large tumours can release multiple cells a day and so this gives it lots of opportunity for cells to affectively metastasise and compromise other organs
