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Molecular Oncology > Day 5: EMT and Transitions between cell states > Flashcards

Day 5: EMT and Transitions between cell states Flashcards

HC13, 14

1
Q

Polarity epithelial cells

A

Apical: towards lumen
Basal: towards lamina basalis and ECM
Lateral: sides

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2
Q

Adherens junction components

A
  • E-cadherin
  • Catenins
  • Actin (cytoskeleton)
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3
Q

EMT in development

A
  • Gastrulation: making mesoderm, and endoderm from epiblast by EMT and then MET for endoderm
    > Primitive streak
  • Neurulation
    > make neural tube and neural crest and top neural tube connected to ectoderm under influence of morphogens like BMP (at dorsal part)
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4
Q

Ectoderm

A

Epithelium and nervous system

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5
Q

EMT concept

A

Anchors between cells dissolved, cell shape changed, apical-basal polarity lost and basement membrane is breached. (basament membrane connected to basal membrane of epithelial cell)

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6
Q

Mesodermal and neural crest cells are both

A

Mesenchymal

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7
Q

Arches of the neural tube and neural crest

A

Differentiation from neural crest part: patterning: to different structures

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8
Q

EMT can be bad: fibrosis and tumor progression

A
  • Fibrosis: epithelial cells become fibroblast like, more motile and deposit scar tissue
  • Tumor progression: break through basement membrane: metastasis (motile)
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9
Q

EMT useful for and not useful in …

A

Useful: developmental and wound healing
Not useful: Fibrosis and tumor progression

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10
Q

Sequence EMT

A

First: loss epithelial features
Next: Acquire mesenchymal features

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11
Q

EMT and MET in metastasis

A

Counterpart is needed (MET)
> cells need to become epithelial again to be able to proliferate and become metastasis > metastatic colonization

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12
Q

Requirements EMT

A
  • Activation EMT Transcription Factors
  • Loss apico-basal polarity
  • E-cadherin repression
  • Invasion and migration
  • Low proliferation (need to be regained with MET)
    > intravasation and extravasation
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13
Q

Counterpart: MET characteristics

A
  • Repression EMT TFs
  • Apico-basal polarity
  • Cell-cell adhesion
  • Increased proliferation
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14
Q

The most fundamental cell state changes in development

A

EMT and MET

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15
Q

Name core TFs orchestrating EMT

A

-Snail (SNAI1)
-Slug (SNAI2)
-Twist
-ZEB1 and ZEB2

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16
Q

SNAIs were discovered in …

A

Drosophila

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17
Q

ZEB1 and ZEB2 involved in development

A

Neurogenesis

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18
Q

Overexpression of Twist leads to gene expression changes:

A
  • Decreased E-cadherin and catenins (alpha, beta, gamma catenin) > epithelial markers
  • Increased mesenchymal markers: Fibronectin, vimentin, N-cadherin, alpha-SMA (smooth muscle actin)
  • b-actin stays similar
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19
Q

Vimentin, N-cadherin, alpha-SMA

A

Vimentin: cytoskeleton
N-cadherin: counterpart E-cadherin
a-SMA: smooth muscle actin

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20
Q

Twist overexpression morphology cells

A

Elongated cells which are motile (ready to move)

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21
Q

Zeb1 promotes EMT and thus … in pancreatic cancer

A

Metastasis

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22
Q

E-cadherin to N-cadherin switch

A
  • E-cadherin binds cells to dissimilar neighbours
  • N-cadherin favors interaction with similar cell types
  • N-cadherin bonds are less restrictive: do not force cells to stick in polarized fashion
    > different cellular interactions
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23
Q

KPC mice (KRAS, P53 and Cre) and states

A

some epithelial (high E-cad/vimentin), some mixed some mesenchymal (low E-cad/vimentin)

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24
Q

KPCZ ice (with Zeb1 KO)

A

EMT driver is KO
> only epithelial
> high E-cad/Vim
> no EMT of cancer cells

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25
Q

Stepwise metastatic cascade: Classic route

A
  • Primary tumor formation
  • Localized invasion
  • Intravasation
  • Transport through circulation
    > hostile environment
    > can interact with platelets
    > travel in clusters to survive with own fibroblasts
  • Arrest in microvessels of various organs
    > typically lung or liver etc
  • Extravasation
    > dependent on invasive and motile character like intravasation
  • Formation micrometastasis
  • Colonization: formation macrometastasis
    > MET: proliferation
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26
Q

Peritoneal metastatic cascade

A
  • Peritoneum metastasis in CRC (or pancreas or liver) or ovarium cancer
  • In peritoneal cavity wall cancer
  • Disseminate directly into peritoneal cavity and survive in environent: not a lot there: some fluid to reduce friction
  • Grow out as metastases: challenging: mesenchymal features needed to survive and reseed
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27
Q

Lymphatic spread (metastatic cascade)

A

-Drains from tissue with maybe tumor
- often targets are LNs

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28
Q

KPCZ mice are … from metastasis

A

Protected
> No Zeb1 for EMT

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29
Q

Proteases in metastasis

A
  • MMPs: matrix metalloproteases which break down ECM
  • Cells are surrounded by other cells and need to move for invasion
  • Break the walls and break basement membrane
    > learned from immune cells which use this
  • Gelatin on microscopy slide can be broken by cancerous cells which express MMPs
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30
Q

Therapy resistance of mesenchymal cells in EPCAM test

A

Sort cells on EPCAM: EPCAM+ is epithelial
> High casp-3: apoptotic
> Epithelial cells after Cisplatin/5-FU treatment: high Casp-3
> Mesenchymal cells: no increase casp-3 after treatment
» resistant to other chemotherapeutics and radiation therapy as well: relatively resistant to apoptosis

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31
Q

KPC Snail KO

A

Rate of metastasis does not go down so much although a EMT TF
> Is EMT needed for metastasis or chemo resistance?
> it is hard to detect cells in primary tumor which have undergone EMT in patients: doubt if EMT is acquired or required for metastasis.

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32
Q

EMT and stemness of cells

A

EMT contributes to the stemness of cells
> limitless proliferative potential
> overexpress Snail: all tumor cells become population of cancer stem cells (also with Twist)
» Facs analysis

33
Q

mRNA profiling cancer stem cells on EMT features

A

Downregulated E-cadherin
Upreguated:
> mesenchymal markers: N-cad, vit, fibronectin
> Zeb2, Twist, Snail, Slug

34
Q

EMT is a key factor for stemness so:

A
  • Cell plasticity
  • Clonogenicity
    (and pluripotency, cells can grow out)
35
Q

KPC mice show more spherical cells seeded than KPCZ mice, so…

A

The EMT KO mice form less spherical (stem-like) cells

36
Q

Uncertain what exactly EMT is required for in cancer cell. Options are:

A

-Mobility
-Resistance
-Stemness

37
Q

Reversability in wound healing

A

Epitheliak > EMT > mesenchymal > MET > epithelial

38
Q

MET happens when …

A

not instructed for EMT

39
Q

Mesenchymal state is …. and … permanent

A

Transient, not permanent
> this reversability is essential for cancer

40
Q

Being mesenchymal comes at cost:

A
  • No proliferation
  • Redundancy
  • Metabolic wiring: different in metabolic state (further than being fibroblast like)
    > to become motile, things are given up like amount of mitochondria
41
Q

TME

A

Extrinsic ques to instruct cancer cells to become mesenchymal (by microenvironment)
> Fibroblasts (not cancerous): paracrine signals
> Macrophages: cytokines and juxtacrine signal

42
Q

Which TME signals

A
  • TGF-beta: induce EMT
    > Feed cells TGFb: decreased E-cad, increased vimentin
    > is reversible: remove TGFb, become epithelial again and proliferate again: repopulate
  • IL-6: immune cytokine and signal for EMT
    > Cells become therapy resistant to standard care and chemo as well
  • Wnt
43
Q

The more TME signals you give …

A

The stronger the EMT phenotype is expressed
> some elements are repressed as well
> first repression epithelial TFs and then induce mesenchymal TFs

44
Q

HC14: Which cells are pluripotent

A

ICM: inner cell mass of developing embryo: contribute to all cell types

45
Q

Which layer contributes to embryonic tissue (layer)

A

epiblast

46
Q

From pluripotency to multipotency

A

ES cells from epiblast to different germ layers and then to different tissue types eventually to monopotency
> lineage restricted at some point
> epigenetically imprinted
> less open chromatin and more DNA methylation
> More lineage specific gene expression

47
Q

Sometimes ability to regain stemness, when

A

Diseases opposite of cancer: when there are too little cells of a cell type
> diabetes, parkinsons, spinal cord disease
» ESC transplantation as option

48
Q

ESC transplantation limitations

A

Not with donors: immune system rejects non self cells
> ethical hurdles of using human embryos

49
Q

Solutions of limitation ESC transplant

A

Human induced pluripotent stem cells (hiPSCs)

50
Q

Induced pluripotency/multipotency

A

Push cells back from differentiated state to intermediate (progenitor) state and maybe even pluripotent state

51
Q

Dolly example

A

Nucleus transplanted from somatic cell to denucleated oocyte: new sheep made: reprogramming to embryonic state
> Some factors in oocyte cytoplasm allows nucleus to be pluripotent
> but in Dolly: still old DNA: short telomeres: development arthritis at young age

52
Q

Yamanaka factors

A

Factors which induce cells to get same effect as nuclear transplant to oocyte
> to make hiPSC
> Used to make from fibroblast (somatic cell)
> c-Myc, Oct4, Sox2, Klf4
> TFs

53
Q

hiPSC procedure and uses

A

Somatic cells isolated from patients > culture > addition Yamanaka factors > selection and expansion of iPS cells > multiple uses
- Disease modelling
- Drug screening
- Patient-specific cell therapy

54
Q

Making the hiPSC TF cocktail

A

Comparison gene expression profiles of stem cells and differentiated cells
> interrogate which are shared genes
> introduce TFs from gene network analysis to differentiated cells

55
Q

First generation hiPSC TF cocktail

A

24 TFs
> stemness was achieved

56
Q

Reprogramming is ….

A

Hard: inefficient and stochastic
> rare occurence of reprogramming in experiment: not right combination of transfected cells

57
Q

Nanog-GFP assay

A

Known gene or TF related with cell state?
> Take bit DNA recognized by NANOG (TF) (binding sequence taken) and put it before GFP (Nanog promotor-GFP assay)
> each cell has this construct
> Only when Nanog activity: green
> Nanog activity when stemness
> Nanog as reporter of stem cells

58
Q

Reduction old TF cocktail for iPS to new

A

From 24 to 4 TFs
> essential TFs for reprogramming identified
> leave one out strategy for experiments
> Oct3/4, Sox2, Klf4, Myc
» Yamanaka factors
» take one away, does not work

59
Q

iPS cells in chimeric animals can contribute to … germ layers

A

all

60
Q

Proof that iPS cells work

A

New mice can be made when iPS cells mixed with ICM in blastocyst with marker
> green marker across all germ layers

61
Q

Early (not used) genetic retroviral mouse model

A

Fertilized oocyte > introduce retroviral elements with information which you want ex vivo and introduced in mouse again
> inject fertilized oocyte with DNA: offspring
not used anymore

62
Q

Chimeric mouse

A

Take ES cells (ES or iPS)
> take transgene in those ES which are well culturable
> inject those in ICM of embryo in mother
> part of the cells of offspring is iPS and some natural ICM

63
Q

Fix the chimeric mouse model

A
  • Gonads are also chimeric: spermatocytes and oocytes
  • cells made from iPS or natural pluripotent cells
  • second generation may have total iPS properties and loss chimeric properties
64
Q

Which cells need to be taken for iPS

A

Almost every cell can be taken
> but, for some more factors needed to add

65
Q

Reprogramming is prevented by …

A

Epigenetic imprinting
> DNA methylation
> Chromatin remodeling

66
Q

Chromatin remodeling

A
  • Histone modifications with PTMs
  • Tightness changed in nucleosomes
  • Acetylation and methylation
67
Q

Epigenetic landscapes

A
  • Normal: transition doable, but boundaries set by others
  • Restrictive: chromatin marks which get cell in some state
  • Permissive: chromatin marks allow for cell type switching or even to pluripotent
68
Q

Nanog-GFP assay with siRNA transfected population > find target > knockdown screen

A

Works well for siRNA against Mbd3 > green colonies formed

69
Q

Mbd3

A

Member of NuRD-complex
> Nucleosome Remodeling and Deacetylase complex: co-repressor complex
> release the epigenetic imprinting marks
> couples histone deacetylase and ATP-dependent chromatin remodeling activities
> level the epigenetic field
> inhibits stemness

70
Q

In epigenetic imprinting, … modifications are important

A

Histone

71
Q

Direct reprogramming

A

Take fibroblasts and reprogram directly to neuronal cells for example instead of via stem cell

72
Q

Direct reprogramming fibroblast to functional neuron

A

Discovered with TauEGFP model: neuronal cells green (Tau marker)
> Factors: Ascl1, Brn2, Myt1L

73
Q

Reprogramming in glioblastoma: marker

A

Surface marker for Tumor-propagating cells (TPCs)
> CD133: stemness marker in glioblastoma and CRC

74
Q

Tumor-propagating cells (TPCs)

A

Small number needed to decrease survival to zero within months in mice
> stemness

75
Q

Distinguish TPCs and DGCs (non-stem cells) based on epigenetics

A

Epigenetic H3K27ac landscape
> results in active DNA: enhancers and promotors
> TPC specific loci where there is H3K27ac (active)

76
Q

Which genes upregulated in TPCs

A

Stem cell markers for pluripotency like Sox2
> epigenetic imprinting leads to stem cell state in TPCs
> these markers are absent in DGCs

77
Q

Introduction stem cell factors in non-stem cells, response in CD133

A

Increased in population that was CD133-
> highly tumorigenic
> different epigentic landscape
> core TPC TFs remodel epigenetic enhancer landscape in DGCs
> set of 4 TFs is sufficient to induce stemness in differentiated glioma cells (DGCs)

78
Q

Cancer and TPCs (in glioblastoma)

A

Cancers contain these tumorigenic stem-like cells which are essential for survival of the tumor

79
Q

Reprogramming in cancer is associated with remodeling of the …

A

Transcriptiome and enhancer landscapes

Molecular Oncology (7 decks)

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