L7-L9 Flashcards
1
Q
EMT/MET are what in cancers?
A
Fatally reactivated in aggressive cancers and other pathologies
2
Q
Define EMT
A
An orchestrated series of events in which cell-cell contacts and cell-ECM contacts are altered to allow release of epithelial cells from surround tissue. The cytoskeleton is reorganised and a new transcriptional program initiated to maintain a mesenhcymal phenotype that supports single cells and collective motility
3
Q
6 characteristics of epithelial cells
A
Typically sheet of 1 cell thick
Cells abutting each other
Regularly spaced adhesions between neighbouring cells
Tight adhesions between cells resulting in lack of movement away from the mono layer
Four types of junctions: gap, tight, adherins, desmosomes which have either apical or basal polarity
All four are lost at some point in EMT
4
Q
Why is epithelium important?
A
Enclosed 3D space
Epithelial sheet is polarised to give different functions and different substrates
The first line of defence against external pathogens
Commonest cancers are epithelial
5
Q
Characteristic of mesenhcymal cells
A
No regimented structure Few intracellular adhesions Weak adhesions for ease of motility Irregular structure Collective migration Extended elongated shape Movement through cell layers like lymphocytes Don't have junctions of adherins. Have integrins, n-cadherins and MMPs to degrade ECM for better migration/ease of migration
6
Q
Failure to close the neural tube due to inapt EMT leads to
A
Spina bifida anencephaly
7
Q
Is EMT reversible?
A
Yes. MET also occurs
8
Q
Epithelial cells have cortical actin what type of actin do mesenhcymal cells have?
A
Actin stress fibres
9
Q
Example of cells that are between Ep/Me states
A
Podocytes in the kidney
10
Q
What gene encodes E-cadherin and how is it repressed in EMT?
A
CDH1 gene encodes E-cad and it is repressed by PRC2 in EMT
11
Q
Define primitively streak
A
A structure that forms in the blastula during early stages of development. It establishes bilateral symmetry the site of gastrulation and initiates germ layer formation.
12
Q
What markers are up regulated in the primitively streak
A
Cripto1
Nodal
Wnt3a
Brachyury
13
Q
Two places EMT occurs in the embryo
A
Gastrulation and Neural crest
14
Q
If E cad is lost by degradation what happens to β catenin?
A
It is not degraded and moves into the nucleus and causes EMT
15
Q
To trigger EMT what TF activates snail and slug?
A
Sox9
16
Q
Example of MET in development?
A
Nephron development
17
Q
Over expression of what
And addition of what gives EMT?
A
Snail and twist you get EMT
Add TGFβ1 you get EMT
18
Q
What is repressed as a result of snail expression?
A
Claudins, occludins, e-cad, desmoplakin
19
Q
Activation of these on snail expression
A
Fibronectin N cadherin Collagen MMPs twist ZEB1 and ZEB2
20
Q
What miRNAs regulate EMT/MET
A
miR34, let7 and miR200
21
Q
In pancreatic and colon adencarcinoma how does snail affect Ecad?
A
Snail binds to CDH1 promoter and physically interacts with PRC2 and SUZ12 to catalyse trimethylation of H3K27 in nearby nucleosomes silencing CDH1
22
Q
What does EMT cause in tumours
A
Tumour initiation
Chemoresistance
Recurrence of tumour
23
Q
GRHL2 can is the only protein that can
A
Sufficiently maintain epithelial phenotype and induce an MET
24
Q
Feedback loops do what to the epithelial and mesenhcymal states?
A
Maintain them
25
SIRT1 is a
Deactylase
26
SUZ12 is a
Methyltransferase
27
Hypothesised roles of EMT in the adult
Generation of adult stem cells
| Maintain tissue homeostasis
28
Confirmed EMT in adults
Pathology ie wound healing
| Cancer
29
What are contextual EC signals that can activate IC EMT factors and where do they come from?
TGFβ, wnt, PDGF5, IL6
| Can be paracrine or autocrine signalling
30
Stages of EMT/MET in cancer progression
Initiation, metastasis, cancer stem cell generation, dormancy and chemoresistance
31
Wnt signalling in cancer
Disregulated in many carcinomas and contributes to expansion and maintenance of CSCs in these tumours.
Hyperactive wnt - aberrant βcat signalling and tumour progression.
32
How may EMT in breast cancer be mediated?
Through snail stabilising wnt activity or transactivation of vimentin by βcat TCF complex
33
In colorectal cancer APC mutation leads to
Constitutive wnt signalling βcat-TCF4 complex binds to ZEB1 promoter and up regulates transcription
34
C-Jun and fra1 are?
Heterodimeric subunits of the AP1 complex stimulated by PDGF5 signalling
35
On TGFβ hyperactivity what happens
It promotes invasion and metastasis in carcinoma and EMT in development
36
In normal and neoplastic tissues EMT is triggered by?
Convergence of multiple signals a cell receives from nearby microenvironment
37
What happens on phosphorylation of SMAD2/3
Forms a complex with SMAD4 and enters the nucleus.
SMAD2/3 associates with certain epigenetics regulators ie TRIM33 which can displace his tone modifications allow chromatin access
38
Mouse hepatocytes response to TGFβ
Reduction of bulk of heterochromatic H3K9me2 marks and increases H3K4me3 and H3K36me3 euchromatic and transcription elongation marks. Gain of activating modifications is crucial for EMT mediated Phenotypes
39
TGFβ can be expressed and cause EMT due to
Oncogenic mutations in ras causing the cell to become more mesenchymal.
40
Ras mutations cause
Epithelial cells to look more mesenchymal (loss of E cad)
41
What are the 10 stages of cancer metastasis?
1. Primary Timor
2. EMT
3. MET - minimal residual disease
4. Local recurrance
5. Intravasation
6. Survival of tumour cells in blood stream
7. Extavasation
8. Micrometastisis
9. Metastasis - tumour cells in two sites
10. Autocrine and paracrine signals at new site influencing surrounding normal cells
42
What markers do CSCs express? And what happens to them ounces they arise?
CD44hi/CD24low
| CSCs may become dormant or form new tumours
43
What may happen to disseminated carcinoma cells at new sites?
They may undergo MET due to lack of maintaining M signals and lapse back into epithelial cells showing the plastic nature of these changes
44
How can disseminated cancer cells survive out of the primary tumour site ie in the circulation?
They are more resistant to environmental and genotoxic stress which is crucial for survival in the circulation
45
EMT signals at the tumour margin gives a partial EMT state. what does this facilitate?
What does full mesenchymal phenotype facilitate?
Mobility and invasion into the stroma. Full mesenchymal phenotype facilitates intravasion and anoikis resistance during dissemination
46
When is best to use epigenetic inhibitors to target progressing cancer cells?
During EMT before CSCs/ differentiated mesenchymal cancer cells develop which can metastasise and are drug resistant
47
How might CSCs maintain mesenchymal traits?
Metastably through the activation of autocrine signalling loops that liberation them from dependence on continuous paracrine MET inductive signals
48
What are active epithelial epigenetic marks?
H3K4me3 and H3Kac
49
What are the chromatin modifying enzymes in MET/EMT?
Polycomb
HDACs
HKDMs
HKMTs
50
How does LSD1 act?
It acts through an amine deoxidise domain which catalyses oxidation of biogenic amines including the N terminus of methylated histones - genetic repression
51
What is the role of LSD1 in EMT/MET?
LSD1 is recruited to epithelial target promoters in snail mediated EMT. It is a lysine de methylated and can cause activation and repression by demethylating H3K4/9. LSD1 can also be recruited by slug and WT1.
52
How are polycomb proteins implicated in EMT?
Repression of epithelial genes by H3K27me3 and recruitment of PRC1.
EZH2 is often over expressed in basal breast cancers and BRCA1 deficient cancers this leads to repression/loss of E-cad. EZH2 is also elevated in aggressive bladder and prostate cancers and there is a correlation with loss of CDH1 expression.
53
How is NuRD implicated in EMT?
NuRD is a complex that contains HDACs ½ dependent on tissue type. It can deacetylate H3K9/14 causing repressive chromatin. Twist can directly associate with NuRD to silence CDH1 in breast cancer
54
How is G9a implicated in EMT?
G9a is a methyltransferase and acts on H3K9me1 (euchromatin) to make H3K9me2. SUV39H is then recruited to the methylated H3 and causes further methylation H3K9me3 and heterochromatic formation. This is mediated by snail and ultimately results in the recruitment of DNMTs which causes promoter methylation of epithelial genes
55
Genetic programs causing cancer in EMT are due to
The transdifferentiation of developmental programs at the wrong times rather than cells becoming neoplastic
56
Three types of EMT
Developmental
Chronic (injury)
Malignant
57
Is EMT direct?
No. Cells can advance to differing extents through an EMT. A spectrum of intermediate Phenotypes
58
What causes the widespread changes in gene expression in EMTs?
Epigenetic modifications causing changes in chromatin structure
59
EMT is a sign of what prognosis?
Poor
60
In what types of breast cancer carcinoma are mesenchymal genes expressed?
Basal and triple negative subtypes of tumour (ER negative, her2 negative and progesterone receptor negative). These cancers have undergone at least 1 partial EMT with some mesenchymal and some epithelial characteristics retained ie quasi state
61
Cancers with quasi state cells what features do they have?
CSC features ie CD44hi and CD24low antigenic state and heightened resistance to diverse cancer therapies as well as enhanced invasive and metastatic properties
62
What can trigger an EMT?
Hypoxia, inflammation, oncogenic stress, metabolic stress
63
When does metastasis occur in cancer?
Fairly early
64
What are the two types of dissemination and what drives one of them?
Early dissemination driven by EMT
| Late dissemination
65
What is wilms tumour an example of?
MET gone wrong. No s shaped body formation.
66
What is the role of WT1 and Wnt4 in nephron development and why do mutations cause wilms tumour?
Wnt4 and WT1 stimulate MET of the metantephric tissue thus preventing formation of the s shaped body.
67
Wilms tumour is (technical term to describe it)
Malignant neoplasm of embryonal nephrogenic elements
68
Three phases of Wilms tumour histopathology
Triphasic:
1 metantephric blastema
2 immature epithelial abortive tubules
3 immature mesenchymal stroma
Unfavourable prog - anaplastic changes rather than triphasic
69
Syndromes associated with WT1
WAGR
Denys-drash syndrome
Beckwidth-wiedeman syndrome
70
Two types of cell origin observed in Wilms tumour
Blastemal-predominant tumour
| StromaL-predominant tumour
71
What happens in blastemal-based wilms tumour?
Unknown mutation of WT1 after induced early mesenchyme and LOI of igf2. Leading to proliferation of early induced mesenchyme and differentiation block. Lack of epithelial differentiation. Nuclear Wt1 remains.
72
What happens in stromal-based wilms tumour?
Primary hit wt1 in uninduced mesenchyme.
2 hit wt1 mutation or LOH and loss of nuclear wt1 in early induced mesenchyme.
Loss of wt1 completely means aberrant differentiation of cells to muscle, fat, cart & bone and stroma.
73
What cells are normally produced in kidney development as a result of MET
Podocytes
Glomerular cells
Tubular epithelial cells
74
What stabilises MYCN to enable proliferation resulting in WT?
Six1/2 dephosphorylates pT58 MYCN (which would normally be degraded). Dephos and stabilisation leads to proliferation and involves tyrosine kinases.
75
What are the best markers for kidney tumours
Six1/2
76
Mutations in unfavourable Wilms tumour
Six1/2 & MYCN - altered mRNA patterns
DROSHA & DGCR8 - impaired miRNA biogenesis
TP53 (p53 mutant) - aneuploidy and chromothripsis
77
How often is DROSHA mutated in wilms tumour?
12%
78
DICER mutations in kidney tumours?
Frequently germline mutations in DICER in renal tumours, but not in sporadic tumours.
So DICER mutations are associated with wilms tumour they are not fully penetrant. So need another hit on DICER or another miRNA gene to enable tumourigenesis
79
Role of lin28 in miRNA biogenesis
Lin28 blocks DICER and therefore expression of nature let7 miRNAs.
80
Mature let7 miRNAs are considered to have?
TSG activity
As seen in HCC - repression of cell growth by miR127 targeting let7
And glioblastoma by miR127 targeting let7 leading to invasion and metastasis in glioblastoma
81
Where is the protocol heron locus?
5q31
82
Where is the MLL locus?
11q24
83
6 diseases associated with Wilms tumour and the wt1 alteration associated
1 WAGR - mostly nonsense
2 DD syndrome mis sense in ZF domain and premature truncations
3 Frazier syndrome splice donor site mutations in intron 9
4 mesothelioma mis sense in the transactivation domain
5 desmoplastic small round cell tumour fusion of Zn fingers 2-4 with end terminus of EWS
6 leukaemias Small insertions on exons 1 and 7
84
Mutations in Wt1 not only cause kidney tumours, but also
Breast, pancreatic and ovarian cancers
85
Knockout wt1 mice
Renal and genital defects and exhibit embryonic lethality
86
Wt1 muts in DD syndrome and Wtum
DD - 90% have wt1 muts
| Wilms tum - 10-20%
87
When Wt1 is over expressed what phenotype occurs
Congenital heart defects
88
Wt1 is an activator and a repressive this is called
Dichotomous transcription factor
89
Some Wt1 fun facts
10 exons
Sense and antisense that overlap exon 1
36 protein isoforms wth different start/stop codons and splicing
Exon 5 can be spliced out
+KTS and -KTS in exon 9
Zn fingers in exons 7,8,9,10
Has a repressive domain and a transactivation domain and a dimerisation domain
90
There are four key wt1 isoforms what are they?
2 with + KTS and 2 with - KTS
91
Wt1 Zn fingers can bind to what and what can inhibit binding ability?
What is Zn held by?
Zn fingers can bind DNA and RNA
Zn can exchange with Cu to block binding ability
Zn is held by CC/HH
92
+KTS wt1 binds to
RNA
93
-KTS wt1 binds with
DNA
94
Where does +KTS bind to with much higher affinity than -KTS?
At speckles (RNA splicing factors)
95
How does wt1 interact with actin?
WT1 binds to actin and is transported to polysomes which is translational machinery.
WT1 appears to be involved in loading RNA onto polysomes. So can regulate at DNA, RNA and protein level
96
Wt1 interacts with what proteins?
BASP1 - via repression domain
CBP - via Zn fingers
WT1 - home dimerisation via NH2 terminus
97
Wt1 binding to CBP causes
Activation of amphiregulin gene by binding the CRE element in the areg promoter
98
KO WT1 in adult mice leads to
```
Death in 9 days
By:
Kidney failure
Pancreas and spleen atrophy
Widespread loss on bone and body fat
RBCs are no longer produced
WT1 is required for adult tissue homeostasis
```
99
Mice lacking the +KTS isoforms show
Decreased cell proliferation of neural progenitor cells. Opposite function to that found in kidneys
100
What is the function of WT1 in the heart?
Wt1 is required for proliferation of vascular progenitors arising from the epicardium and maintenance of an undifferentiated state.
In the developing heart Wt1 also seems to be needed for EMT.
101
How does βcat link to WT1 in the kidney and in the heart?
βcat is activated in wt1 mutant tumours. This is not linked by wnt 4 (no wnt4 in mesenchymal active tissues).
Activated β cat can force cells into premature EMT. (EMT processes in the heart are linked to canonical wnt signalling)
Showing contrast between wt1 in the urogenital system and other systems
102
Wt1/βcat interactions seem to...
Regulate the mesenchymal epithelial balance
103
What is rosa26?
A locus used for constitutive ubiquitous gene expression in mice. Exhibits a broad lacZ staining in haemopoietic and tissue cells. Useful for chimera analysis
104
Loss of Wt1 on E cad?
Differentiation and suppression
105
What drops with wt1 is knockout?
Igf1
106
What cells is wt1 expressed in all of?
Mesothelium of all cells
107
What is visceral fat?
Fat stored within the abdominal cavity around all organs. Not subcutaneous fat which is beneath the skin
108
Wt1 has what relationship with visceral fat?
It is expressed in all visceral fat pads. It's expression is not detected in subcutaneous and BAT.
109
How does wt1 control EMT?
Through snail and suppression of Ecadherin
110
Wt1 cause epicardial ES cells to undergo EMT into what cardiac cells?
Those important for myovasculature
111
After myocardial infection what happens to Wt1?
Wt1 is temporarily up regulated in endothelial cells of the I fat yet tea and the boarder zone of the heart
112
How does wt1 affect wnt4 to cause an MET in the kidney and EMT the heart on a chromatin level?
In the epicardium wt1 assocs with the co-repressor BASP1 causing the chromatin to be repressed
In the kidney WT1 does not associate with BASP1 instead assocs with the co-activator CBP/p300 to activate wnt4 which stimulates MET
113
Where does wt1 +co-act/rep bind in on the wnt4 locus in the kidney and epicardium?
In the same place to WREs (wt1 response elements) Wt1 loss in epicardium lead to ectopic expression of wnt4 by the same wt1 regulatory elements. Suggesting that wt1 spatially regulates wnt4 expression bidirectionally in kidney vs epicardium
