Day 1: Introduction CRC, Wnt signalling pathway and targets, Stem cell development Flashcards
HC 01, 02, 03, 04
1
Q
TNM staging in CRC: T staging (tumor size)
A
T1: Tumor only in inner layer of bowel
T2: Tumor into muscle layer of bowel wall
T3: Tumor has grown into outer lining of bowel wall
T4: Tumor has grown through outer lining and may have grown into other structures
2
Q
TNM staging CRC: N and M staging (lymph nodes and metastasis)
A
N0: No lymph nodes containing cancer cells
N1: 1-3 lymph nodes close to the bowel contain cancer cells
N2: 4+ lymph nodes close to the bowel contain cancer cells
M0: No metastasis
M1: Metastases
3
Q
Cancer stages and CRC treatment strategies
A
Other kind of staging for overall tumor
- Stage 1: Surgery
- Stage 2: Surgery and sometimes adj. Chemo
- Stage 3: Surgery and Chemo
- Stage 4: Chemo and targeted therapy and sometimes surgery (bad prognosis)
4
Q
Histology plate with cancer with a lot of stroma means?
A
More chance of metastasis
5
Q
Different cancers and relative expression in character?
A
Can differ
6
Q
How long does it take to develop adenomas in colon? And cancer?
A
5-20 years for adenoma
From there: 5-15 years to cancer
10-35 years until cancer total
7
Q
Why does the development of the cancer take so long?
A
Multi-hit model
8
Q
Carcinogenesis promotion by SV40 virus
A
Contains LT which inhibits p53 and RB in the cell (cell cycle regulators)
> loss cell cycle control for short time
> induce senescence programs after short period of unregulated cell proliferation
> fast exponential growth is then stopped
9
Q
How is senescence induced in rapid proliferating cells?
A
Short telomers
10
Q
Gene and protein for extending telomers in stem cells and mutated cells
A
Telomerase (in human: hTERT gene)
> a proto-oncogene, should not get gain-of-function
> used in stem cells
11
Q
LT + TERT hits in cells (mutations/gains)
A
Longer proliferation than only LT, but still stops
> control regained
12
Q
LT + TERT mutation + Ras mutation
A
Can grow in vitro and in vivo
> Ras mutation > classic oncogene
13
Q
Ras mutation
A
Ras induces signalling for proliferation
> mutation: continuously active
14
Q
How many mutations in CRC (colorectal cancer)?
A
1500-1.5 mutations
> majority mutations is for non-functional DNA (however, noncoding has functions)
15
Q
Six Hallmarks of Cancer
A
-Self-sufficiency in growth signals
-Insensitive to antigrowth signals
-Tissue invasion and metastasis
-Limitless replicative potential
-Sustained angiogenesis
-Resist apoptosis
16
Q
Telomerase mutation: for which hallmark of cancer?
A
Limitless replicative potential
17
Q
Ras mutation hallmark
A
Self-sufficiency in growth signals
18
Q
Vogelgram for CRC
A
Normal epithelium
> Loss of APC
Hyperplastic epithelium
>
Early adenoma
> Activation KRAS
Intermediate adenoma
> Loss Smad4
Late adenoma
> Loss p53
Carcinoma
> invasion
Invasive metastasis
19
Q
How many hit model is the Vogelgram for CRC?
A
4-hit model
20
Q
4-hit model order of mutations. Is this rigid
A
APC>KRAS>Smad4>p53
-It can vary
-When early inflammation: p53 before APC
21
Q
Ras mutation and 17p loss (p53 mutation) in early, intermediate and late adenoma
A
Early: not a lot of Ras mutation and 17p loss
Intermediate: a lot of Ras mutation but not a lot of 17p loss
Late: both high
> first Ras mutation, then p53 loss
22
Q
Familial adenomatous polyposis (FAP) and gene involved
A
-On early age: development many adenomas in colon (benign)
-100% penetrance of colon cancer: develops always
> other malignancies can develop
-APC locus involved
> Chromosome 5 mutation
23
Q
Juvenile polyposis and gene involved
A
-Juvenile polyp formation (adenomas)
-Familial syndrome: early onset
-Caused when loss BMP signalling
> Mutation BMPR1 gene
> Mutation SMAD4 gene
24
Q
BMP signalling result for colon cancer development
A
Induce differentiation in intestine: block colon cancer growth
25
The 4 hits from the 4-hit-model and the related Hallmarks of cancer
- APC: Self-sufficiency in growth signals
- KRAS: Self-sufficiency in growth signals
- SMAD4: Insensitivity to anti-growth signals
- P53: Evading apoptosis
26
4-hit-model and in vitro colon cancer in organoids
-APC CRISPR mutation, and also P53, KRAS and SMAD4 mutations induced.
> only growth when all 4 mutations in organoids
> three-fold mutation: fast proliferation which is halted later
27
What characteristics (name, not list) does cancer need to be called that?
Hallmarks of cancer
28
HC02: Identification INT1
Retroviral-induced cancer > insertion DNA and overactive promotor near oncogene to make them overactive > more expression oncogene and other nearby genes
> Common integration site in retroviral-induced cancer: INT1
29
Retroviral life cycle
Infection cell with RNA
> Reverse transcription
> Insertion into chromosomal DNA
> Transcription and translation > assembly and budding of the virus.
30
Drosophila born without wings
Defect in the Wg (wingless) gene > defect in segmentation (polarity) and development
> same phenotype with deficient Wg and Tcf
31
The INT1 region and Wg gene both relate to the gene
Wnt
32
How many types of Wnt genes
Around 17
33
Functions Wnt
-Development and stem cell biology
-Cell proliferation
34
Where expression Wnt?
Wnt has different specialized functions, in tissues for neuronal development, segementation, proliferation
35
Where is Wnt located in the 4-hit model (the Vogelgram)?
First hit: at the loss of APC
36
Result loss of APC
Formation adenomas > hyperproliferation
37
CRC patients without APC mutation mostly have a mutated ...
beta-catenin
38
APC mutations and Beta-catenin mutations occur ...
Mutually exclusive
> hardly co-occur
> same pathway: no advantage for both mutated (no selective pressure in cancer development)
39
Reasons for mutually exclusive mutations
-Same pathway
-Toxic together which leads to cell death
40
Discovery Beta-catenin target
TCF-1 (a TF highly expressed in T-lymphocytes and important for intestinal epithelium)
> Luciferase expression test, TCF-b-catenin complex activates expression and fluorescence.
41
Which part of APC is mutated in cancer?
b-catenin binding domain
42
Blocking beta-catenin pathway which is active in cancer in vitro by addition:
APC is lost in cancer
> add wild-type (WT) APC > no expression Luciferase
43
APC is an inhibitor of ...
b-catenin induced transcription
44
APC mutation in cancer
No inhibition of beta-catenin: no more control
> continuous b-catenin-TCF induced transcrption
> gene programs for cell proliferation
> hyperproliferation
45
APC binding function, and what if this is disrupted?
APC can bind b-catenin
> truncation of b-catenin at binding region for APC > no inhibition of the b-catenin-TCF transcription program: hyperproliferation.
46
Mutations of beta-catenin
On specific locations on N-terminal serines or threonines
> No phosphorylation of b-catenin
> target of kinases, regulation b-catenin
47
Which kinases can phosphorylate b-catenin
GSK3b and CK1
> on N-terminal residues
> not in TCF binding site (or APC binding site ofc)
> different role in regulating b-catenin
48
Effect high expression of Wnt
Wnt signalling: APC blocked (destruction complex blocked), b-catenin released to bind TCF and activate target genes
49
Lynch syndrome (HNPCC) and often mutation
Genetic predisposition for colon cancer
> often mutated b-catenin
50
Receptors for Wnt
Frizzled and Lrp
51
Wnt binding to receptors response
Receptors transduces signal to disassemble destruction complex for beta-catenin with the kinases in them
52
Destruction complex b-catenin
B-catenin
APC: Interaction with b-catenin
GSK3b and CK: kinases for b-cat
Axin: scaffold for complex
> repressed state when complex formed
53
Effect phosphorylation b-catenin
Targeting for polyubiquitination and destruction in proteasome
> binding N-terminal residue phosphate groups to an E3 ubiquitin ligase
54
Function RNF43
Transmembrane E3 ligase
> polyubiquitinate receptor for Wnt (Frizzled)
> Breakdown Frizzled
> Lower signal when Wnt present and bound. (because less receptors present, less activation Wnt pathway)
55
Regulation RNF43
A ligand: R-spondin (RSPO) can bind extracellularly to RNF43 and induces membrane clearance of membrane bound RNF43
> R-spondin binds RNF43 and LGR5 (transmembrane receptor, induces membrane clearance)
> Wnt + R-sponding: local signal for proliferation: high signal
56
What happens if RNF43 is no longer expressed or active?
Wnt ligands overstimulate pathway
57
Repres Wnt signalling when RNF43 deletion
Inhibit Wnt primary signal
58
Why does blocking Wnt primary signal not work in treatment?
APC mutated: upstream signalling not involved, b-catenin always active
59
HC03: Where are cells formed and where do they die in intestinal lining
Death in tops villus and formation in crypt base
60
Phases of intestinal epithelium along crypt and villus from crypt to villus top
Proliferation > differentiation > apoptosis
61
Where Wnt signalling in intestine
Bottom of crypts
> upregulation proliferation
> Wnt signalling essential for stem cells
62
Result APC-/- mice in intestine
Formation adenomas (Wnt signalling overactive)
63
Effect APC deletion of differentiation intestinal cells
Inhibited: hyperproliferation leads to less differentiation
64
TCF deletion effect in intestine
No activation target genes Wnt signallin, b-catenin and TCF complex cannot bind DNA: no proliferation intestinal epithelium (no villi)
> less food uptake
> death because no enterocyte formation
65
Finding downstream targets of Wnt signalling
-RNA sequencing
-Compare APC mutation with WT
-Differential activity genes
> 350 downregulated genes
> 250 upregulated genes
66
Downregulated and upregulated genes when CRC cell lines with inactive Wnt (dnTCF, no TCF)
Downregulated genes: Crypt genes, TCF targets (interesting)
Upregulated genes: villus genes (differentiation), (not so interesting)
67
Wnt targets are ... expressed in adenomas
Highly
68
In adenomas, classic Wnt targets are more present (overexpressed), including Axin, Fzd (Frizzled) and Myc. Although Axin inhibits Wnt signalling. Explain.
It is a target of Wnt signalling > auto-regulation Wnt pathway
> not a problem for the cancer: APC deficient or b-catenin mutated
69
Relevance of downstream targets Wnt pathway
Crucial targets for CRC!
> should be expressed at right location: base of crypts
70
Markers crypt bases (Wnt downstream targets)
-Ascl2
-Lgr5
71
Mouse technology: transgenic mice
-gene of interest active from embryonal phase
> everywhere in mouse (disadvantage) > solution: include promotor (promotor insertion) which is selective for activation in certain tissue: only in colon for example
72
Result Ascl2 transgenic mice
Crypt expansion
> hyperproliferation from birth
> Ascl2 brought in with intestinal promotor
> bad food absorption: proliferation overrules differntiationL no enterocytes, mice die
73
Ascl2 in Wnt pathway
Wnt binds receptors
> Inhibition APC
> APC inhibits b-cat (which binds TCF)
> B-cat with TCF activates expression Ascl2
> Ascl2 promotes transcription for proliferation
74
More advanced mouse technology: Cre DNA recombinase
Cre DNA recombinase (from bacteriophage) recognizes specific loxP sites in DNA > cuts at these sites > deletion of genes induced
- Cre: constitutively active recombinase
- Piece between loxP sites is cleaved and DNA deleted
75
CreER
Inducible recombinase which responds to hormone tamoxifen (activation after addition)
> Induce deletion of genes after normal development of mice: not like the normal transgenic mice
> not affected by dysregulation Wnt pathway until tamoxifen added
> CreER gene inserted at place of other gene like Lgr5 : take over the Wnt promotor. Expressed when Wnt pathway active and activated when tamoxifen
76
Dis/Advantages transgenic mouse tech with CreER
-No control of insertion site
-Expression controlled by promotor (cell type specific) and Cre
-Cre can be expressed constitutively or inducible
> cell specificity and inducibility
77
CreER with LacZ transgenic mouse technology for detecting Wnt signalling
CreER inserted at place Lgr5
> Lgr5 promotor taken over: Wnt pathway
> CreER expressed when Wnt
> Induced with tamoxifen
> CreER cuts out premature stop sequence before LacZ gene
> LacZ expression and detection
78
Myc function and path in pathway
Downstream target Wnt
> expression via B-cat/TCF
> Induces transcription for proliferation
79
Result Villin-CreER APC and Myc KO mice
APC and Myc KO (deletion) can be induced with CreER which is added to Villin promotor (replaced for Villin)
> Villin: intestine specific promotor
> Tamoxifen: deletion APC > overactive Wnt pathway: hyperproliferation
> Deletion Myc: no hyperproliferation when deletion APC: normalization of situation
80
Effect APC mutation in Vogelgram (to which stage)
Hyperplastic epithelium: formation adenomas
81
Ratio APC and b-cat mutation
80%: APC mutation
10%: b-cat mutation
Other 10% has overactive Wnt pathway as well: essential for CRC
82
What kind of molecule and gene is Wnt?
-Ligand
-Oncogene: overactive expression promotes hyperproliferation
-Morphogen: for regulation of segmentation in Drosphila for example
83
What kind of molecule is Ascl2
A transcription factor
84
HC04: What kind of cell is a stem cell
Non-specialized cell
85
Where do you find stem cells?
In virtually all organs
86
Characteristics stem cells
-Non-specialized
-Can divide asymmetrically (stem cell vs progenitor)
-Unlimited division capacity
-Generate specific subset of differentiated cells (when multipotent)
87
Omnipotent stem cells
Fertilized oocyte
88
Why do stem cells have limitless replicative capacity?
Telomerase activity
89
Why stem cells needed?
Regeneration of cells because cells die
90
Side effects chemotherapy and radiotherapy and role stem cells
Hair loss and mucositis
> after therapy: rapid regeneration: stem cells resistant against therapy.
91
Are pools of stem cells limited?
Yes
92
Hemopoietic stem cells characteristic
> limited amounts and essential for survival: a lot of regeneration in body
93
Give Mice hematopoietic stem cell KO hematopoietic progenitor cells effect
No long-term survival
> stem cells exhausted: the progenitor cells are no long-living stem cells
94
Where stem cells in intestinal epithelium
Between Paneth cells in base of crypts
95
Function villi in intestines (including microvilli)
Increase surface for absorption
96
Stem cell fates in intestinal crypts
-Absorpative cell: enterocytes: for nutrients and fluids
-Secretory cells
> Goblet cell: mucus
> Enteroendocrine cell: hormones to blood
> Paneth cell: for integrity stem cells and immunity (does not move up, one month survival): granules with antimicrobial peptides and immunomodulating proteins.
97
Where do the enterocytes take up the most fluid?
In the colon
98
Definition stem cell
Non-specialized cell that posses capacity to self-renew and to generate specific set of heterogeneous cell lineages that make up a tissue
99
Method to prove that a cell is a stem cell
Lineage tracking
> mark stem cells
> progenitors also marked by differentiation
> differentiated cells also marked
> if you trace progenitors: not long term repopulation, the label will disappear (no self renewal)
> self renewal required (lon term repopulation)
100
Morphogens which regulate homeostasis pathways in intestinal lining
Wnt, Bmp, Notch
101
Functions of morphogens in differentiation in intestinal epithelium
-Wnt: proliferation
-BMP: induction differentiation
-Notch: direction differentiation
102
What does Wnt regulate in crypt?
Proliferation of cells
> Too much upregulated: hyperproliferation
> Downregulated: no differentiation as villi, unrepairable damage
103
The best stem cell marker for lineage tracing in crypt
Lgr5 (one of the Wnt target genes, to inhibit Rnf43 after bining R-spondin).
104
Lineage tracing using ROSA26
Make CreER with GFP at Lgr5 gene so that it is placed after promotor
> expression when Wnt upregulated
> Activation with Tamoxifen
> When no tamoxifen: green fluorescence
> Rosa26 promotor inserted as well: promotor which is active in all cells with a Stop region before LacZ gene (blue)
> Tamoxifen: active CreER: stop region cut out > transcription LacZ > Blue color (with green)
- You can see in intestinal lining that the cells start (and keep!) colouring blue gradually from crypt to villus top.
105
Organoids for crypt research
Mini organ system cultured in lab
> isolated crypt dmains from donor
> formation mini organ with crypt and villus domains
> also used for tumor tissues
106
Organoid research discovered an essential interaction for intestinal stem cells, namely:
Lgr5+ stem cells interact with Paneth cells
> decreased colony forming efficiency when Lgr5 doublets or singlet
>> but when given Wnt ligands
> no colony formaiton of Paneth singlet
> better proliferation when interaction: Paneth cells are source of Wnt signalling molecule
107
Environmental ligands for Wnt signalling made around stem cells in crypt
Wnt by Paneth cells
R-spondin by fibroblasts around base crypt
108
BMP effects
Blocks crypt proliferation and allows for differentiation
109
Which cells secrete BMP
Mesenchyme
110
Mutation in BMP signalling at which levels?
In the BMPR1 (receptor, together with BMPR2) or Smad4 (intracellular transducer)
111
Which molecule level regulates BMP signalling?
Noggin
112
Effect BMP on Wnt signalling
BMP inhibits Wnt signalling: stop proliferation: possibility to differentiate
113
Where expression BMP
Everywhere in intestinal epithelium
114
Where production and secretion Noggin and concentration when hyperproliferation/adenoma
In bottom of crypt
> Noggin inhibits function BMP signalling and pathway
> Wnt can be active: proliferation and stemness
> when hyperproliferation: Overexpression Noggin and repression BMP
115
How is crypt expansion regulated in BMP signalling level
Mutant BMPR1 or Smad4
Noggin overexpression
116
Notch characteristics
- Transmembrane receptor in cells crypt
- Ligands are expressed in crypt as well (transmembrane delta; contact dependent signalling)
- Notch receivers differentiation into secretory lineage (goblet, enteroendocrine, Paneth)
- Essential for stem cell maintenance
117
Lateral inhibition with Notch
Cell with more Delta than Notch: Goblet differentiation: downregulation Notch production and upregulation Delta
> receiving surrounding cells bind Delta/jagged with Notch and become absorptive cells (enterocytes): downregulation Delta
118
What happens if you would delete the stem cells from the crypts?
New stem cells are generated
119
Over- and underexpression Notch effects
Overexpression: only enterocytes
Inhibition: only secretory cells
120
Cells at top villus
Apoptosis and release: debris gobbled up for reuse nutrients
121
Back-up system stem cells in crypt
Other cells become stem cells
> cells get back into stem cell niche
122
Bmi1 marking with CreER / Rosa26 staining with Lgr5+GFP
No effect, green cells as stem cells but no blue
> but combine with deletion and death of all Lgr5+ stem cells: effect as stem cells
> Bmi1+ cells become new stem cells when stem cells depleited
> in the lining from base to top crypt: Fast cycling repopulating stem cell and differenitated Paneth cell/niche -- slow cycling/resting.back-up stem cell (Bmi1+) -- fast cycling progenitor cells -- differentiated cells
