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Molecular Oncology > Day 1: Introduction CRC, Wnt signalling pathway and targets, Stem cell development > Flashcards

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

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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

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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)

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4
Q

Histology plate with cancer with a lot of stroma means?

A

More chance of metastasis

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5
Q

Different cancers and relative expression in character?

A

Can differ

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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

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7
Q

Why does the development of the cancer take so long?

A

Multi-hit model

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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

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9
Q

How is senescence induced in rapid proliferating cells?

A

Short telomers

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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

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11
Q

LT + TERT hits in cells (mutations/gains)

A

Longer proliferation than only LT, but still stops
> control regained

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12
Q

LT + TERT mutation + Ras mutation

A

Can grow in vitro and in vivo
> Ras mutation > classic oncogene

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13
Q

Ras mutation

A

Ras induces signalling for proliferation
> mutation: continuously active

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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)

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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

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16
Q

Telomerase mutation: for which hallmark of cancer?

A

Limitless replicative potential

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17
Q

Ras mutation hallmark

A

Self-sufficiency in growth signals

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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

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19
Q

How many hit model is the Vogelgram for CRC?

A

4-hit model

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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

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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

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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

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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

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24
Q

BMP signalling result for colon cancer development

A

Induce differentiation in intestine: block colon cancer growth

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25
Q

The 4 hits from the 4-hit-model and the related Hallmarks of cancer

A
  • APC: Self-sufficiency in growth signals
  • KRAS: Self-sufficiency in growth signals
  • SMAD4: Insensitivity to anti-growth signals
  • P53: Evading apoptosis
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26
Q

4-hit-model and in vitro colon cancer in organoids

A

-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

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27
Q

What characteristics (name, not list) does cancer need to be called that?

A

Hallmarks of cancer

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28
Q

HC02: Identification INT1

A

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

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29
Q

Retroviral life cycle

A

Infection cell with RNA
> Reverse transcription
> Insertion into chromosomal DNA
> Transcription and translation > assembly and budding of the virus.

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30
Q

Drosophila born without wings

A

Defect in the Wg (wingless) gene > defect in segmentation (polarity) and development
> same phenotype with deficient Wg and Tcf

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31
Q

The INT1 region and Wg gene both relate to the gene

A

Wnt

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32
Q

How many types of Wnt genes

A

Around 17

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33
Q

Functions Wnt

A

-Development and stem cell biology
-Cell proliferation

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34
Q

Where expression Wnt?

A

Wnt has different specialized functions, in tissues for neuronal development, segementation, proliferation

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35
Q

Where is Wnt located in the 4-hit model (the Vogelgram)?

A

First hit: at the loss of APC

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36
Q

Result loss of APC

A

Formation adenomas > hyperproliferation

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37
Q

CRC patients without APC mutation mostly have a mutated …

A

beta-catenin

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38
Q

APC mutations and Beta-catenin mutations occur …

A

Mutually exclusive
> hardly co-occur
> same pathway: no advantage for both mutated (no selective pressure in cancer development)

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39
Q

Reasons for mutually exclusive mutations

A

-Same pathway
-Toxic together which leads to cell death

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40
Q

Discovery Beta-catenin target

A

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.

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41
Q

Which part of APC is mutated in cancer?

A

b-catenin binding domain

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42
Q

Blocking beta-catenin pathway which is active in cancer in vitro by addition:

A

APC is lost in cancer
> add wild-type (WT) APC > no expression Luciferase

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43
Q

APC is an inhibitor of …

A

b-catenin induced transcription

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44
Q

APC mutation in cancer

A

No inhibition of beta-catenin: no more control
> continuous b-catenin-TCF induced transcrption
> gene programs for cell proliferation
> hyperproliferation

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45
Q

APC binding function, and what if this is disrupted?

A

APC can bind b-catenin
> truncation of b-catenin at binding region for APC > no inhibition of the b-catenin-TCF transcription program: hyperproliferation.

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46
Q

Mutations of beta-catenin

A

On specific locations on N-terminal serines or threonines
> No phosphorylation of b-catenin
> target of kinases, regulation b-catenin

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47
Q

Which kinases can phosphorylate b-catenin

A

GSK3b and CK1
> on N-terminal residues
> not in TCF binding site (or APC binding site ofc)
> different role in regulating b-catenin

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48
Q

Effect high expression of Wnt

A

Wnt signalling: APC blocked (destruction complex blocked), b-catenin released to bind TCF and activate target genes

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49
Q

Lynch syndrome (HNPCC) and often mutation

A

Genetic predisposition for colon cancer
> often mutated b-catenin

50
Q

Receptors for Wnt

A

Frizzled and Lrp

51
Q

Wnt binding to receptors response

A

Receptors transduces signal to disassemble destruction complex for beta-catenin with the kinases in them

52
Q

Destruction complex b-catenin

A

B-catenin
APC: Interaction with b-catenin
GSK3b and CK: kinases for b-cat
Axin: scaffold for complex
> repressed state when complex formed

53
Q

Effect phosphorylation b-catenin

A

Targeting for polyubiquitination and destruction in proteasome
> binding N-terminal residue phosphate groups to an E3 ubiquitin ligase

54
Q

Function RNF43

A

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
Q

Regulation RNF43

A

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
Q

What happens if RNF43 is no longer expressed or active?

A

Wnt ligands overstimulate pathway

57
Q

Repres Wnt signalling when RNF43 deletion

A

Inhibit Wnt primary signal

58
Q

Why does blocking Wnt primary signal not work in treatment?

A

APC mutated: upstream signalling not involved, b-catenin always active

59
Q

HC03: Where are cells formed and where do they die in intestinal lining

A

Death in tops villus and formation in crypt base

60
Q

Phases of intestinal epithelium along crypt and villus from crypt to villus top

A

Proliferation > differentiation > apoptosis

61
Q

Where Wnt signalling in intestine

A

Bottom of crypts
> upregulation proliferation
> Wnt signalling essential for stem cells

62
Q

Result APC-/- mice in intestine

A

Formation adenomas (Wnt signalling overactive)

63
Q

Effect APC deletion of differentiation intestinal cells

A

Inhibited: hyperproliferation leads to less differentiation

64
Q

TCF deletion effect in intestine

A

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
Q

Finding downstream targets of Wnt signalling

A

-RNA sequencing
-Compare APC mutation with WT
-Differential activity genes
> 350 downregulated genes
> 250 upregulated genes

66
Q

Downregulated and upregulated genes when CRC cell lines with inactive Wnt (dnTCF, no TCF)

A

Downregulated genes: Crypt genes, TCF targets (interesting)
Upregulated genes: villus genes (differentiation), (not so interesting)

67
Q

Wnt targets are … expressed in adenomas

A

Highly

68
Q

In adenomas, classic Wnt targets are more present (overexpressed), including Axin, Fzd (Frizzled) and Myc. Although Axin inhibits Wnt signalling. Explain.

A

It is a target of Wnt signalling > auto-regulation Wnt pathway
> not a problem for the cancer: APC deficient or b-catenin mutated

69
Q

Relevance of downstream targets Wnt pathway

A

Crucial targets for CRC!
> should be expressed at right location: base of crypts

70
Q

Markers crypt bases (Wnt downstream targets)

A

-Ascl2
-Lgr5

71
Q

Mouse technology: transgenic mice

A

-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
Q

Result Ascl2 transgenic mice

A

Crypt expansion
> hyperproliferation from birth
> Ascl2 brought in with intestinal promotor
> bad food absorption: proliferation overrules differntiationL no enterocytes, mice die

73
Q

Ascl2 in Wnt pathway

A

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
Q

More advanced mouse technology: Cre DNA recombinase

A

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
Q

CreER

A

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
Q

Dis/Advantages transgenic mouse tech with CreER

A

-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
Q

CreER with LacZ transgenic mouse technology for detecting Wnt signalling

A

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
Q

Myc function and path in pathway

A

Downstream target Wnt
> expression via B-cat/TCF
> Induces transcription for proliferation

79
Q

Result Villin-CreER APC and Myc KO mice

A

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
Q

Effect APC mutation in Vogelgram (to which stage)

A

Hyperplastic epithelium: formation adenomas

81
Q

Ratio APC and b-cat mutation

A

80%: APC mutation
10%: b-cat mutation
Other 10% has overactive Wnt pathway as well: essential for CRC

82
Q

What kind of molecule and gene is Wnt?

A

-Ligand
-Oncogene: overactive expression promotes hyperproliferation
-Morphogen: for regulation of segmentation in Drosphila for example

83
Q

What kind of molecule is Ascl2

A

A transcription factor

84
Q

HC04: What kind of cell is a stem cell

A

Non-specialized cell

85
Q

Where do you find stem cells?

A

In virtually all organs

86
Q

Characteristics stem cells

A

-Non-specialized
-Can divide asymmetrically (stem cell vs progenitor)
-Unlimited division capacity
-Generate specific subset of differentiated cells (when multipotent)

87
Q

Omnipotent stem cells

A

Fertilized oocyte

88
Q

Why do stem cells have limitless replicative capacity?

A

Telomerase activity

89
Q

Why stem cells needed?

A

Regeneration of cells because cells die

90
Q

Side effects chemotherapy and radiotherapy and role stem cells

A

Hair loss and mucositis
> after therapy: rapid regeneration: stem cells resistant against therapy.

91
Q

Are pools of stem cells limited?

A

Yes

92
Q

Hemopoietic stem cells characteristic

A

> limited amounts and essential for survival: a lot of regeneration in body

93
Q

Give Mice hematopoietic stem cell KO hematopoietic progenitor cells effect

A

No long-term survival
> stem cells exhausted: the progenitor cells are no long-living stem cells

94
Q

Where stem cells in intestinal epithelium

A

Between Paneth cells in base of crypts

95
Q

Function villi in intestines (including microvilli)

A

Increase surface for absorption

96
Q

Stem cell fates in intestinal crypts

A

-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
Q

Where do the enterocytes take up the most fluid?

A

In the colon

98
Q

Definition stem cell

A

Non-specialized cell that posses capacity to self-renew and to generate specific set of heterogeneous cell lineages that make up a tissue

99
Q

Method to prove that a cell is a stem cell

A

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
Q

Morphogens which regulate homeostasis pathways in intestinal lining

A

Wnt, Bmp, Notch

101
Q

Functions of morphogens in differentiation in intestinal epithelium

A

-Wnt: proliferation
-BMP: induction differentiation
-Notch: direction differentiation

102
Q

What does Wnt regulate in crypt?

A

Proliferation of cells
> Too much upregulated: hyperproliferation
> Downregulated: no differentiation as villi, unrepairable damage

103
Q

The best stem cell marker for lineage tracing in crypt

A

Lgr5 (one of the Wnt target genes, to inhibit Rnf43 after bining R-spondin).

104
Q

Lineage tracing using ROSA26

A

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
Q

Organoids for crypt research

A

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
Q

Organoid research discovered an essential interaction for intestinal stem cells, namely:

A

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
Q

Environmental ligands for Wnt signalling made around stem cells in crypt

A

Wnt by Paneth cells
R-spondin by fibroblasts around base crypt

108
Q

BMP effects

A

Blocks crypt proliferation and allows for differentiation

109
Q

Which cells secrete BMP

A

Mesenchyme

110
Q

Mutation in BMP signalling at which levels?

A

In the BMPR1 (receptor, together with BMPR2) or Smad4 (intracellular transducer)

111
Q

Which molecule level regulates BMP signalling?

A

Noggin

112
Q

Effect BMP on Wnt signalling

A

BMP inhibits Wnt signalling: stop proliferation: possibility to differentiate

113
Q

Where expression BMP

A

Everywhere in intestinal epithelium

114
Q

Where production and secretion Noggin and concentration when hyperproliferation/adenoma

A

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
Q

How is crypt expansion regulated in BMP signalling level

A

Mutant BMPR1 or Smad4
Noggin overexpression

116
Q

Notch characteristics

A
  • 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
Q

Lateral inhibition with Notch

A

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
Q

What happens if you would delete the stem cells from the crypts?

A

New stem cells are generated

119
Q

Over- and underexpression Notch effects

A

Overexpression: only enterocytes
Inhibition: only secretory cells

120
Q

Cells at top villus

A

Apoptosis and release: debris gobbled up for reuse nutrients

121
Q

Back-up system stem cells in crypt

A

Other cells become stem cells
> cells get back into stem cell niche

122
Q

Bmi1 marking with CreER / Rosa26 staining with Lgr5+GFP

A

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

Molecular Oncology (7 decks)

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