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DG & EC > L7-L9 > Flashcards

L7-L9 Flashcards

1
Q

EMT/MET are what in cancers?

A

Fatally reactivated in aggressive cancers and other pathologies

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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

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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

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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

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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
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6
Q

Failure to close the neural tube due to inapt EMT leads to

A

Spina bifida anencephaly

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7
Q

Is EMT reversible?

A

Yes. MET also occurs

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8
Q

Epithelial cells have cortical actin what type of actin do mesenhcymal cells have?

A

Actin stress fibres

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9
Q

Example of cells that are between Ep/Me states

A

Podocytes in the kidney

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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

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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.

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12
Q

What markers are up regulated in the primitively streak

A

Cripto1
Nodal
Wnt3a
Brachyury

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13
Q

Two places EMT occurs in the embryo

A

Gastrulation and Neural crest

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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

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15
Q

To trigger EMT what TF activates snail and slug?

A

Sox9

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16
Q

Example of MET in development?

A

Nephron development

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17
Q

Over expression of what

And addition of what gives EMT?

A

Snail and twist you get EMT

Add TGFβ1 you get EMT

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18
Q

What is repressed as a result of snail expression?

A

Claudins, occludins, e-cad, desmoplakin

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19
Q

Activation of these on snail expression

A 
Fibronectin 
N cadherin 
Collagen MMPs
twist 
ZEB1 and ZEB2
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20
Q

What miRNAs regulate EMT/MET

A

miR34, let7 and miR200

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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

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22
Q

What does EMT cause in tumours

A

Tumour initiation
Chemoresistance
Recurrence of tumour

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23
Q

GRHL2 can is the only protein that can

A

Sufficiently maintain epithelial phenotype and induce an MET

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24
Q

Feedback loops do what to the epithelial and mesenhcymal states?

A

Maintain them

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25
Q

SIRT1 is a

A

Deactylase

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26
Q

SUZ12 is a

A

Methyltransferase

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27
Q

Hypothesised roles of EMT in the adult

A

Generation of adult stem cells

Maintain tissue homeostasis

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28
Q

Confirmed EMT in adults

A

Pathology ie wound healing

Cancer

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29
Q

What are contextual EC signals that can activate IC EMT factors and where do they come from?

A

TGFβ, wnt, PDGF5, IL6

Can be paracrine or autocrine signalling

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30
Q

Stages of EMT/MET in cancer progression

A

Initiation, metastasis, cancer stem cell generation, dormancy and chemoresistance

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31
Q

Wnt signalling in cancer

A

Disregulated in many carcinomas and contributes to expansion and maintenance of CSCs in these tumours.
Hyperactive wnt - aberrant βcat signalling and tumour progression.

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32
Q

How may EMT in breast cancer be mediated?

A

Through snail stabilising wnt activity or transactivation of vimentin by βcat TCF complex

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33
Q

In colorectal cancer APC mutation leads to

A

Constitutive wnt signalling βcat-TCF4 complex binds to ZEB1 promoter and up regulates transcription

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34
Q

C-Jun and fra1 are?

A

Heterodimeric subunits of the AP1 complex stimulated by PDGF5 signalling

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35
Q

On TGFβ hyperactivity what happens

A

It promotes invasion and metastasis in carcinoma and EMT in development

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36
Q

In normal and neoplastic tissues EMT is triggered by?

A

Convergence of multiple signals a cell receives from nearby microenvironment

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37
Q

What happens on phosphorylation of SMAD2/3

A

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

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38
Q

Mouse hepatocytes response to TGFβ

A

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

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39
Q

TGFβ can be expressed and cause EMT due to

A

Oncogenic mutations in ras causing the cell to become more mesenchymal.

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40
Q

Ras mutations cause

A

Epithelial cells to look more mesenchymal (loss of E cad)

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41
Q

What are the 10 stages of cancer metastasis?

A
  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
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42
Q

What markers do CSCs express? And what happens to them ounces they arise?

A

CD44hi/CD24low

CSCs may become dormant or form new tumours

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43
Q

What may happen to disseminated carcinoma cells at new sites?

A

They may undergo MET due to lack of maintaining M signals and lapse back into epithelial cells showing the plastic nature of these changes

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44
Q

How can disseminated cancer cells survive out of the primary tumour site ie in the circulation?

A

They are more resistant to environmental and genotoxic stress which is crucial for survival in the circulation

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45
Q

EMT signals at the tumour margin gives a partial EMT state. what does this facilitate?
What does full mesenchymal phenotype facilitate?

A

Mobility and invasion into the stroma. Full mesenchymal phenotype facilitates intravasion and anoikis resistance during dissemination

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46
Q

When is best to use epigenetic inhibitors to target progressing cancer cells?

A

During EMT before CSCs/ differentiated mesenchymal cancer cells develop which can metastasise and are drug resistant

47
Q

How might CSCs maintain mesenchymal traits?

A

Metastably through the activation of autocrine signalling loops that liberation them from dependence on continuous paracrine MET inductive signals

48
Q

What are active epithelial epigenetic marks?

A

H3K4me3 and H3Kac

49
Q

What are the chromatin modifying enzymes in MET/EMT?

A

Polycomb
HDACs
HKDMs
HKMTs

50
Q

How does LSD1 act?

A

It acts through an amine deoxidise domain which catalyses oxidation of biogenic amines including the N terminus of methylated histones - genetic repression

51
Q

What is the role of LSD1 in EMT/MET?

A

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
Q

How are polycomb proteins implicated in EMT?

A

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
Q

How is NuRD implicated in EMT?

A

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
Q

How is G9a implicated in EMT?

A

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
Q

Genetic programs causing cancer in EMT are due to

A

The transdifferentiation of developmental programs at the wrong times rather than cells becoming neoplastic

56
Q

Three types of EMT

A

Developmental
Chronic (injury)
Malignant

57
Q

Is EMT direct?

A

No. Cells can advance to differing extents through an EMT. A spectrum of intermediate Phenotypes

58
Q

What causes the widespread changes in gene expression in EMTs?

A

Epigenetic modifications causing changes in chromatin structure

59
Q

EMT is a sign of what prognosis?

A

Poor

60
Q

In what types of breast cancer carcinoma are mesenchymal genes expressed?

A

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
Q

Cancers with quasi state cells what features do they have?

A

CSC features ie CD44hi and CD24low antigenic state and heightened resistance to diverse cancer therapies as well as enhanced invasive and metastatic properties

62
Q

What can trigger an EMT?

A

Hypoxia, inflammation, oncogenic stress, metabolic stress

63
Q

When does metastasis occur in cancer?

A

Fairly early

64
Q

What are the two types of dissemination and what drives one of them?

A

Early dissemination driven by EMT

Late dissemination

65
Q

What is wilms tumour an example of?

A

MET gone wrong. No s shaped body formation.

66
Q

What is the role of WT1 and Wnt4 in nephron development and why do mutations cause wilms tumour?

A

Wnt4 and WT1 stimulate MET of the metantephric tissue thus preventing formation of the s shaped body.

67
Q

Wilms tumour is (technical term to describe it)

A

Malignant neoplasm of embryonal nephrogenic elements

68
Q

Three phases of Wilms tumour histopathology

A

Triphasic:
1 metantephric blastema
2 immature epithelial abortive tubules
3 immature mesenchymal stroma

Unfavourable prog - anaplastic changes rather than triphasic

69
Q

Syndromes associated with WT1

A

WAGR
Denys-drash syndrome
Beckwidth-wiedeman syndrome

70
Q

Two types of cell origin observed in Wilms tumour

A

Blastemal-predominant tumour

StromaL-predominant tumour

71
Q

What happens in blastemal-based wilms tumour?

A

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
Q

What happens in stromal-based wilms tumour?

A

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
Q

What cells are normally produced in kidney development as a result of MET

A

Podocytes
Glomerular cells
Tubular epithelial cells

74
Q

What stabilises MYCN to enable proliferation resulting in WT?

A

Six1/2 dephosphorylates pT58 MYCN (which would normally be degraded). Dephos and stabilisation leads to proliferation and involves tyrosine kinases.

75
Q

What are the best markers for kidney tumours

A

Six1/2

76
Q

Mutations in unfavourable Wilms tumour

A

Six1/2 & MYCN - altered mRNA patterns
DROSHA & DGCR8 - impaired miRNA biogenesis
TP53 (p53 mutant) - aneuploidy and chromothripsis

77
Q

How often is DROSHA mutated in wilms tumour?

A

12%

78
Q

DICER mutations in kidney tumours?

A

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
Q

Role of lin28 in miRNA biogenesis

A

Lin28 blocks DICER and therefore expression of nature let7 miRNAs.

80
Q

Mature let7 miRNAs are considered to have?

A

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
Q

Where is the protocol heron locus?

A

5q31

82
Q

Where is the MLL locus?

A

11q24

83
Q

6 diseases associated with Wilms tumour and the wt1 alteration associated

A

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
Q

Mutations in Wt1 not only cause kidney tumours, but also

A

Breast, pancreatic and ovarian cancers

85
Q

Knockout wt1 mice

A

Renal and genital defects and exhibit embryonic lethality

86
Q

Wt1 muts in DD syndrome and Wtum

A

DD - 90% have wt1 muts

Wilms tum - 10-20%

87
Q

When Wt1 is over expressed what phenotype occurs

A

Congenital heart defects

88
Q

Wt1 is an activator and a repressive this is called

A

Dichotomous transcription factor

89
Q

Some Wt1 fun facts

A

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
Q

There are four key wt1 isoforms what are they?

A

2 with + KTS and 2 with - KTS

91
Q

Wt1 Zn fingers can bind to what and what can inhibit binding ability?
What is Zn held by?

A

Zn fingers can bind DNA and RNA
Zn can exchange with Cu to block binding ability
Zn is held by CC/HH

92
Q

+KTS wt1 binds to

A

RNA

93
Q

-KTS wt1 binds with

A

DNA

94
Q

Where does +KTS bind to with much higher affinity than -KTS?

A

At speckles (RNA splicing factors)

95
Q

How does wt1 interact with actin?

A

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
Q

Wt1 interacts with what proteins?

A

BASP1 - via repression domain
CBP - via Zn fingers
WT1 - home dimerisation via NH2 terminus

97
Q

Wt1 binding to CBP causes

A

Activation of amphiregulin gene by binding the CRE element in the areg promoter

98
Q

KO WT1 in adult mice leads to

A 
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
Q

Mice lacking the +KTS isoforms show

A

Decreased cell proliferation of neural progenitor cells. Opposite function to that found in kidneys

100
Q

What is the function of WT1 in the heart?

A

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
Q

How does βcat link to WT1 in the kidney and in the heart?

A

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
Q

Wt1/βcat interactions seem to…

A

Regulate the mesenchymal epithelial balance

103
Q

What is rosa26?

A

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
Q

Loss of Wt1 on E cad?

A

Differentiation and suppression

105
Q

What drops with wt1 is knockout?

A

Igf1

106
Q

What cells is wt1 expressed in all of?

A

Mesothelium of all cells

107
Q

What is visceral fat?

A

Fat stored within the abdominal cavity around all organs. Not subcutaneous fat which is beneath the skin

108
Q

Wt1 has what relationship with visceral fat?

A

It is expressed in all visceral fat pads. It’s expression is not detected in subcutaneous and BAT.

109
Q

How does wt1 control EMT?

A

Through snail and suppression of Ecadherin

110
Q

Wt1 cause epicardial ES cells to undergo EMT into what cardiac cells?

A

Those important for myovasculature

111
Q

After myocardial infection what happens to Wt1?

A

Wt1 is temporarily up regulated in endothelial cells of the I fat yet tea and the boarder zone of the heart

112
Q

How does wt1 affect wnt4 to cause an MET in the kidney and EMT the heart on a chromatin level?

A

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
Q

Where does wt1 +co-act/rep bind in on the wnt4 locus in the kidney and epicardium?

A

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

DG & EC (6 decks)

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