Day 5: EMT and Transitions between cell states Flashcards
HC13, 14
Polarity epithelial cells
Apical: towards lumen
Basal: towards lamina basalis and ECM
Lateral: sides
Adherens junction components
- E-cadherin
- Catenins
- Actin (cytoskeleton)
EMT in development
- 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)
Ectoderm
Epithelium and nervous system
EMT concept
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)
Mesodermal and neural crest cells are both
Mesenchymal
Arches of the neural tube and neural crest
Differentiation from neural crest part: patterning: to different structures
EMT can be bad: fibrosis and tumor progression
- Fibrosis: epithelial cells become fibroblast like, more motile and deposit scar tissue
- Tumor progression: break through basement membrane: metastasis (motile)
EMT useful for and not useful in …
Useful: developmental and wound healing
Not useful: Fibrosis and tumor progression
Sequence EMT
First: loss epithelial features
Next: Acquire mesenchymal features
EMT and MET in metastasis
Counterpart is needed (MET)
> cells need to become epithelial again to be able to proliferate and become metastasis > metastatic colonization
Requirements EMT
- 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
Counterpart: MET characteristics
- Repression EMT TFs
- Apico-basal polarity
- Cell-cell adhesion
- Increased proliferation
The most fundamental cell state changes in development
EMT and MET
Name core TFs orchestrating EMT
-Snail (SNAI1)
-Slug (SNAI2)
-Twist
-ZEB1 and ZEB2
SNAIs were discovered in …
Drosophila
ZEB1 and ZEB2 involved in development
Neurogenesis
Overexpression of Twist leads to gene expression changes:
- 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
Vimentin, N-cadherin, alpha-SMA
Vimentin: cytoskeleton
N-cadherin: counterpart E-cadherin
a-SMA: smooth muscle actin
Twist overexpression morphology cells
Elongated cells which are motile (ready to move)
Zeb1 promotes EMT and thus … in pancreatic cancer
Metastasis
E-cadherin to N-cadherin switch
- 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
KPC mice (KRAS, P53 and Cre) and states
some epithelial (high E-cad/vimentin), some mixed some mesenchymal (low E-cad/vimentin)
KPCZ ice (with Zeb1 KO)
EMT driver is KO
> only epithelial
> high E-cad/Vim
> no EMT of cancer cells
Stepwise metastatic cascade: Classic route
- 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
Peritoneal metastatic cascade
- 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
Lymphatic spread (metastatic cascade)
-Drains from tissue with maybe tumor
- often targets are LNs
KPCZ mice are … from metastasis
Protected
> No Zeb1 for EMT
Proteases in metastasis
- 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
Therapy resistance of mesenchymal cells in EPCAM test
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
KPC Snail KO
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.
EMT and stemness of cells
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
mRNA profiling cancer stem cells on EMT features
Downregulated E-cadherin
Upreguated:
> mesenchymal markers: N-cad, vit, fibronectin
> Zeb2, Twist, Snail, Slug
EMT is a key factor for stemness so:
- Cell plasticity
- Clonogenicity
(and pluripotency, cells can grow out)
KPC mice show more spherical cells seeded than KPCZ mice, so…
The EMT KO mice form less spherical (stem-like) cells
Uncertain what exactly EMT is required for in cancer cell. Options are:
-Mobility
-Resistance
-Stemness
Reversability in wound healing
Epitheliak > EMT > mesenchymal > MET > epithelial
MET happens when …
not instructed for EMT
Mesenchymal state is …. and … permanent
Transient, not permanent
> this reversability is essential for cancer
Being mesenchymal comes at cost:
- 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
TME
Extrinsic ques to instruct cancer cells to become mesenchymal (by microenvironment)
> Fibroblasts (not cancerous): paracrine signals
> Macrophages: cytokines and juxtacrine signal
Which TME signals
- 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
The more TME signals you give …
The stronger the EMT phenotype is expressed
> some elements are repressed as well
> first repression epithelial TFs and then induce mesenchymal TFs
HC14: Which cells are pluripotent
ICM: inner cell mass of developing embryo: contribute to all cell types
Which layer contributes to embryonic tissue (layer)
epiblast
From pluripotency to multipotency
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
Sometimes ability to regain stemness, when
Diseases opposite of cancer: when there are too little cells of a cell type
> diabetes, parkinsons, spinal cord disease
» ESC transplantation as option
ESC transplantation limitations
Not with donors: immune system rejects non self cells
> ethical hurdles of using human embryos
Solutions of limitation ESC transplant
Human induced pluripotent stem cells (hiPSCs)
Induced pluripotency/multipotency
Push cells back from differentiated state to intermediate (progenitor) state and maybe even pluripotent state
Dolly example
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
Yamanaka factors
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
hiPSC procedure and uses
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
Making the hiPSC TF cocktail
Comparison gene expression profiles of stem cells and differentiated cells
> interrogate which are shared genes
> introduce TFs from gene network analysis to differentiated cells
First generation hiPSC TF cocktail
24 TFs
> stemness was achieved
Reprogramming is ….
Hard: inefficient and stochastic
> rare occurence of reprogramming in experiment: not right combination of transfected cells
Nanog-GFP assay
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
Reduction old TF cocktail for iPS to new
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
iPS cells in chimeric animals can contribute to … germ layers
all
Proof that iPS cells work
New mice can be made when iPS cells mixed with ICM in blastocyst with marker
> green marker across all germ layers
Early (not used) genetic retroviral mouse model
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
Chimeric mouse
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
Fix the chimeric mouse model
- 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
Which cells need to be taken for iPS
Almost every cell can be taken
> but, for some more factors needed to add
Reprogramming is prevented by …
Epigenetic imprinting
> DNA methylation
> Chromatin remodeling
Chromatin remodeling
- Histone modifications with PTMs
- Tightness changed in nucleosomes
- Acetylation and methylation
Epigenetic landscapes
- 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
Nanog-GFP assay with siRNA transfected population > find target > knockdown screen
Works well for siRNA against Mbd3 > green colonies formed
Mbd3
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
In epigenetic imprinting, … modifications are important
Histone
Direct reprogramming
Take fibroblasts and reprogram directly to neuronal cells for example instead of via stem cell
Direct reprogramming fibroblast to functional neuron
Discovered with TauEGFP model: neuronal cells green (Tau marker)
> Factors: Ascl1, Brn2, Myt1L
Reprogramming in glioblastoma: marker
Surface marker for Tumor-propagating cells (TPCs)
> CD133: stemness marker in glioblastoma and CRC
Tumor-propagating cells (TPCs)
Small number needed to decrease survival to zero within months in mice
> stemness
Distinguish TPCs and DGCs (non-stem cells) based on epigenetics
Epigenetic H3K27ac landscape
> results in active DNA: enhancers and promotors
> TPC specific loci where there is H3K27ac (active)
Which genes upregulated in TPCs
Stem cell markers for pluripotency like Sox2
> epigenetic imprinting leads to stem cell state in TPCs
> these markers are absent in DGCs
Introduction stem cell factors in non-stem cells, response in CD133
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)
Cancer and TPCs (in glioblastoma)
Cancers contain these tumorigenic stem-like cells which are essential for survival of the tumor
Reprogramming in cancer is associated with remodeling of the …
Transcriptiome and enhancer landscapes
