9. Invasion and Metastasis Flashcards
1
Q
what proportion of mortality is caused by metastasis?
A
90%
2
Q
what is the major site of metastasis for colon cancer?
A
liver
3
Q
do all circulating cancer cells form metastasis? why?
A
only some will form metastases because there are many steps that must occur
3
Q
6 major steps in metastasis
A
- invasion and infiltration of surrounding normal host tissue
- release of neoplastic cells
- survival in circulation
- arrest in capillary beds of distant organs
- penetration of lymphatic or blood vessel walls
- growth of disseminated tumour cells
4
Q
5 main changes for metastasis
A
- cell detachment
- invasion of stroma
- intravasation
- migration
- extravasation
5
Q
5 things that occur for cell detachment
A
- cells acquire spindle shape for migration
- increased matrix-degrading proteinases
- increased growth factors in original and metastatic site
- decreased adhesion molecules
- decreased proteinase inhibitors
6
Q
2 requirements for cells as they migrate
A
- resist immune cells in circulation
- anchor themselves via increased endothelial cell adhesion molecules
7
Q
3 molecules upregulated for extravasation
A
- selectin ligands
- integrins
- matrix-degrading proteinases
8
Q
what 2 things increase for metastases?
A
- increased cell-cell adhesion molecules
- increased growth factors
9
Q
what ultimately determines the fate of metastasis?
A
microenvironment of primary and distant site
10
Q
6 in vivo experimental models
A
- surgical biopsies
- histopathology, “omics”, RNAseq
- animal tumour models
- transgenic/KO/knockin mice
- patient-derived xenograft
- circulating tumour cells
11
Q
what do histopathology, “omics”, and RNAseq tell us?
A
shows what primary tumour cells acquire/suppress to be able to grow at secondary site
12
Q
4 types of animal tumour models
A
- induction by chemicals, oncogenic viruses
- transplantation
- spontaneous metastasis
- experimental metastasis
13
Q
what is spontaneous metastasis in animal model?
A
put kidney tumour cells directly into kidney to see path of metastasis
14
Q
what is experimental metastasis in animal model?
A
put kidney tumour cells in organ of later stages
15
Q
describe patient-derived xenograft
A
immediately put patient cells in animal to keep tumour cells as close to origin as possible
16
Q
how do we get circulating tumour cells?
A
aka liquid biopsy –> isolate cells in circulation that have shed from tumour
17
Q
5 in vitro experimental models
A
- cell lines
- reconstituted tissue
- ECM models
- genetically altered cells
- PDX, organoids, slices
18
Q
what happens at the primary site at beginning of invasion? (4)
A
- hyperplasia
- loss of polarity
- loss of tissue organization
- intact basement membrane
19
Q
what happens during microinvasion?
A
proteolytic enzymes (MMP2, MMP9) break the basement membrane and cells begin migration
20
Q
what does EMT stand for?
A
Epithelial –> Mesenchymal Transition
21
Q
what does EMT allow for?
A
allows for cells to be motile
22
Q
3 things that happen to cells during EMT?
A
- Lose organization
- Cell-cell contacts are disrupted
- Cells become immortalized
23
Q
5 indications of EMT
A
- E-cadherin DOWNregulated
- Epithelial integrins DOWNregulated
- N-cadherin UPregulated
- Vimentin UPregulated
- Fibronectin UPregulated
24
what is the role of E-cadherin?
part of junctional complex holding epithelial cells together
25
what is the role of epithelial integrins?
receptor for basement membrane proteins
26
what is vimentin?
cytoskeletal protein unique to mesenchymal cells
27
what is the role of fibronectin?
migration track for cells
28
why do cells become spindle-shaped?
lets them migrate along fibronectin matrix
29
4 cellular adhesion molecules
1. E-cadherin
2. Ig superfamily with Ig domain (N-CAM)
3. Mucin-like CAM
4. Integrins
30
role of mucin-like CAM
binds selectins
normally expressed on blood vessels and immune cells
31
why does a metastasizing tumor cell express selectin?
so it can bind mucin-like CAM on blood vessels to allow for migration
32
what do integrins bind (3)
1. ECM
2. N-CAMs
3. cadherins
33
3 roles of integrins
1. adhesion
2. polarity
3. migration
34
what 2 CAMs have homophilic interactions?
1. E-cadherin
2. N-CAM
35
what 2 CAMs have heterophilic interactions?
1. Mucin-like CAM
2. Integrins
36
describe E-cadherin as a hallmark of EMT
DECREASED E-cadherin is a hallmark of EMT
37
assay showing decreased E-cadherin in EMT
induce EMT with Akt expression (Akt downstream of growth factor receptors)
before Akt (aka before EMT): E-cadherin is at cell boundary for cell-cell attachment
after Akt (aka after EMT): E-cadherin diffused thru cytoplasm so no cell-cell attachment
38
5 types of cadherins and locations
1. Neural (neurons, muscle, endothelial cells)
2. Epithelial
3. Placental
4. Retinal
5. Endothelial
39
6 functions of cadherins
1. mediate Ca2+ HOMOTYPIC cell-cell adhesion
2. mediate cell sorting during embryogenesis
3. form cellular junctions to form tissues so cells recognize each other
4. establish cell polarity
5. inhibit apoptosis
6. activate/inhibit growth factor receptors
40
how do cadherins allow cells to recognize each other?
ex. cells expressing E-selectin will seek out other cells expressing E-selectin
41
how do cadherins change cell shape? (4 steps)
1. extracellular domain binds p120
2. p120 is stabilized at the membrane
3. beta-catenin and alpha-catenin are recruited
4. alpha-catenin binds actin to allow stretching of cells to give specific cell shape
42
cadherins act as a bridge btwn:
cadherins act as a bridge btwn cytoskeleton and extracellular environment
43
describe beta-catenin and its downstream function
pre-assembled in ER and acts as transcription factor for Wnt signaling --> important for development and embryogenesis
44
what do HAV-containing peptides do?
disrupt adhesion and trigger cell migration
45
describe cadherins and cancer
A. well-differentiated, poorly invasive adenomas have HIGH E-cadherin
B. invasive carcinomas have LOW E-cadherins
46
what causes reduced E-cadherin levels in invasive carcinomas? (4)
1. transcriptional repression/inactivation
2. DNA methylation
3. mutations
4. post-translational modification
47
what makes E-cadherin de-regulated?
phosphorylation of E-cadherin, beta-catenin, p120 by RTK
48
3 things that reduced E-cadherin leads to?
1. increased cell motility
2. increases invasion
3. triggers beta-catenin/lymphoid enhancer binding factor (LEF1) regulated transcription
49
2 opposite roles of beta-catenin
1. keeps cells attached via cadherin complex
2. if E-cadherin decreases, beta-catenin is released and goes to nucleus to act as TF and cause EMT
50
how do integrins bind ECM?
via glycoproteins
51
2 types of glycoproteins
1. fibronectins
2. laminins
52
what is the ECM? its role?
web of proteins and carbohydrates at cell surface --> connect the cell exterior to cytoskeletal fibres on interior
53
how are integrins expressed?
always expressed as a complex, each with unique ligand specificity
54
what are beta3, 5, 6, and 8 integrin subunits for?
endothelial cell receptors that mediate angiogenesis
55
what does alpha5beta1 bind?
what does alpha2beta1 bind?
what does alpha6beta4 bind?
alpha5beta1 --> fibronectin
alpha2beta1 --> collagen
alpha6beta4 --> laminin
56
what is the first step of integrin signaling called?
INSIDE-OUT signaling
57
describe INSIDE-OUT signaling for integrin signaling (activation)
1. integrin starts out inactive --> folded over PM and binds ECM proteins with low affinity
2. chemokines and growth factors activate integrin --> elongated and 2 cytoplasmic domains separate to form pocket for ECM to bind with high affinity
58
what does activation of integrin require?
talin
59
function of talin
recruited due to GPCR into integrin beta-subunit and begins process for elongation and separation
60
describe OUTSIDE-IN signaling (3 steps)
1. once ECM binds activated integrin, there is clustering and creation of focal adhesions
2. signaling proteins, tyrosine kinases, focal adhesion kinases recruited
3. signaling begins!
61
relationship btwn integrins and actin
integrins communicate with actin polymerase to change cell shape
62
3 steps of integrin + actin relationship
1. inactive talin activated by GPCR or RTK
2. talin forms dimer which binds and activates integrins
3. integrins mediate actin polymerization
63
role of RTK?
mediate EMT and involved in diff stages of metastasis
64
role of VEGFR1 and VEGFR3
VEGFR1 = driver of angiogenesis
VEGFR3 = driver of lymph angiogenesis
65
unique thing about INSR/IGF1R
Only RTK family that is expressed on surface as DIMER --> when ligand binds, there is conformational change in cytoplasmic domain where 1 kinase domain can phosphorylate tyrosine in the other subunit
66
6 steps of RTK
1. Growth factor binds
2. Receptor dimerizes
3. Receptor auto-phosphorylates
4. Adapter proteins and GEF protein bind
5. Ras-GTP activates protein kinase cascade
6. TFs become phosphorylated to alter gene expression
67
4 results of mutations in RTK
1. Autocrine loop
2. Amplification
3. Constitutive activation
4. Genomic rearrangement (constitutively dimerized)
68
what does EGFR activate? (2)
how does it relate to integrin?
1. Akt for cell survival and metabolism
2. Erk for cell proliferation
more potent than integrin
69
when does EMT occur?
during normal development and cancer
70
2 key regulators of EMT program
Snail1 and Snail2
71
4 roles of Snail signaling
1. Link Wnt, TGFbeta, notch signaling
2. Upregulate MMP proteinases
3. Upregulate Lef-1 partner of beta-catenin
4. Block E-cadherin transcription
72
what upregulates Snail signaling?
NFkB
73
What does beta-catenin do once Snail signaling causes E-cadherin to decrease?
when there's reduced E-cadherin, beta catenin will activate transcription
74
role of GSK3B
Guardian of beta-catenin stability --> keeps beta-catenin levels low by phosphorylating it and targeting it for degradation
75
how do RTK and integrins affect GSK3B
Activate Akt by phosphorylation to inactivate GSK3B
76
2 results of inactivated GSK3B
1. Prevents beta-catenin degradation --> allows it to accumulate for increased transcription
2. activates Snail1/2
77
overall, 2 ways beta-catenin increase transcription for EMT
1. Reduced E-cadherin = increased beta-catenin
2. RTK and integrins inactivate GSK3B
78
3 ways that TGFbeta induces EMT
1. GSK3B
2. SMAD2/3
3. aPKC
^all upregulate Snail to repress E-cadherin
79
what happens to cells treated with TGFbeta
cells become mesenchymal-like --> induces stress fibers and stretching
80
in addition to EMT, what is an effect of TGFbeta?
regulates tumour microenvironment --> macrophage polarization, blocking T cell response to tumour
81
what is cadherin switching? what induces it (2)?
switches from E to N cadherin --> induced by TGFbeta and beta-catenin
82
where is N-cadherin normally expressed? how does this help the migrating cells?
N-cadherin is normally expressed on endothelium and nerve cells
epithelial cells lose ability to recognize each other so now they can bind endothelial and nerve cells at N-cadherin for migration
83
in addition to physically helping migrating cells move, 2 other functions of cadherin switching
1. N-cadherin stabilizes growth factor receptors
2. N-cadherin competes with E-cadherin for p120 so beta-catenin can be released into cytoplasm
84
N-cadherin and growth factor receptors (3)
1. Stabilizes FGFR to drive cell proliferation and migration
2. Stabilizes PDGFR to drive cell motility
3. E-cadherin normally inhibits IGF, EGF, and FGF but N-cadherin releases this inhibition
85
3 overall results of cadherin switching?
1. increased detachment
2. increased motility
3. increased survival
86
3 pieces of evidence for cadherin switching
1. N-cadherin and other non-typical cadherins found in many tumours
2. Non-metastatic carcinoma cell can become metastatic upon N-cadherin overexpression
3. Carcinoma cells become non-invasive and non-metastatic upon N-cadherin silencing
87
how do we know that cadherin switching is a LATE event?
Transgenic expression of N-cadherin does not induce tumourigenesis and does not alter tumour onset --> therefore not oncogenic, but helps once cancer has developed
88
3 models of tumour migration
1. Mesenchymal --> migrate in mesenchymal way as single or multi cell
2. Solid strand --> migrate as cluster of cells
3. Outward pushing tumour --> push thru as solid mass to break down tissue
89
4 parts of single cell migration
1. rear-end retraction
2. actomyosin contraction
3. proteolysis at leading edge
4. pseudopod production, adhesion, traction force, proteolysis to break ECM
90
collective cell migration
LEADING EDGE (aka microtrack) --> some cells make a channel to pull the rest of the cells along
Macrotrack --> the rest of the cells induce ECM remodeling along interface
91
3 requirements of invasion
1. tumour cells go thru many tissue compartments made of cells that are separated by ECM
2. secretion of destructive enzymes that can locally degrade ECM
3. tissue architecture breaks down so tumour can expand, invade blood vessels, and spread to distant sites
92
what stages of metastasis require invasion?
ALL
93
5 stages of metastasis that require invasion
1. development of invasive potential
2. expansive growth + basement membrane invasion
3. angiogenesis, intravasation, transport thru body
4. arrest and extravasation at secondary site
5. invasion of secondary tissue for micro/macrometastasis
94
what are MMPs regulated by? (2)
beta catenin and SNAIL
95
what 2 ions do MMPs rely on?
1. Zn2+
2. Ca2+
96
normal physiologic function of MMPs?
for matrix degradation when there is turnover of tissue where basement membrane must be broken
97
4 main subclasses of MMPs
1. collagenases
2. gelatinases
3. stromelysins
4. MT-MMPs
98
regulation of MMPs during metastasis
normally, MMPs have on/off signals for regulation but this system is broken during metastasis
99
are most MMPs secreted?
yes, except MT-MMPs
100
basic structure of MMP
1. signal peptide
2. pro domain
3. catalytic domain
101
role of pro domain
covers active site to prevent MMP from degrading matrix when not needed
102
what is unique about gelatinases? why?
have fibronectin-like domains
integrins bind fibronectin so if there's integrins in focal adhesion, MMP can bind these sites and concentrate themselves for a lot of proteoly
103
2 examples of gelatinases
MMP2 and MMP9
104
what is unique about MT-MMPs?
have unique transmembrane domain that allows them to sit on PM and concentrate proteolysis in 1 spot on cell surface
105
4 ways of MMP regulation
1. gene expression
2. activation
3. inhibition
4. proteolytic cascades and feedback mechanisms
106
what is the mechanism for MMP activation called? how does it work (3 steps)?
called CYSTEINE-SWITCH
1. pro domain is cleaved/chemical enters
2. Zn within active domain has cysteine replaced by water
3. Active site is no longer sequestered and enzyme can begin breaking down ECM
107
3 roles of MT-MMPs?
1. act as MMPs for proteolysis
2. on cell surface can cleave pro domain to activate other MMPs
3. on cell surface can cause shedding of molecules like TNF
108
what are invadopodia?
actin-rich protrusions of PM associated with ECM degradation
109
what are found in invadopodia? (2)
1. mediators like RTK
2. MMPs
110
4 stages of invadopodia formation
1. TRIGGER --> integrin, chemokine, growth factor
2. forms scaffold that accumulate in site of actin polymerization and induce actin polymerization
3. MMP14/19 recruited to stabilize scaffold
4. MMPs activated to degrade matrix in focalized site for invadopodia maturation
111
what do we expect to happen to invasion and invadopodia with KO chemokine receptor?
less invasion, less invadopodia
112
why is invadopodia an efficient way to invade?
concentrates all degradation power in 1 place
113
ADAMs and ADAMTs
- type of molecule
- transmembrane or secreted?
ADAM = disintegrin and MMP (transmembrane)
ADAMT = disintegrin and MMP with thrombospondin motifs (secreted)
114
when are ADAMs and ADAMTs highly expressed?
in cancers
115
7 domains of ADAM
1. Pro domain
2. MMP domain
3. Disintegrin domain
4. EGF domain
5. cysteine-rich domain
6. transmembrane domain
7. cytoplasmic domain
116
why are ADAMs and ADAMTs aka sheddases?
can cleave PM proteins to secrete ectodomains
117
role of ADAM and ADAMT
for ECM proteolysis, EMT, etc
118
3 ADAM substrates
1. degrade E-cadherin
2. TNFalpha
3. Notch
119
4 non-specific MMP inhibitors + where are they produced and found?
1. Alpha2-macroglobulin
2. Alpha1-proteinase inhibitor
3. Alpha1-chymotrypsin
4. Alpha2-antiplasmin
produced in liver, circulate in plasma
120
specific MMP inhibitors
- how are they produced?
- NT domain?
- CT domain?
- where do they act?
TIMPs 1-4
- produced by tumour or host cells from diff genes
- NT domain = 125 aa
- CT domain = 65 aa
- act regionally/specifically
121
how do TIMPs 1-4 interact with MMPs (2)?
INACTIVE MMP --> bind pro domain to block activation
ACTIVE MMP --> blocks catalytic activity
122
why is the tumour microenvironment important for proteolytic cascades? (2)
1. MMPs activate each other
2. other enzymes, like plasmin, can activate MMPs
123
4 factors that the microenvironment can produce for angiogenesis
1. VEGF-A
2. VEGF-C
3. Pro-tumour macrophages
4. Immune cells, tumour cell, TNFalpha, integrins
124
role of VEGF-A in angiogenesis
initiates angiogenesis from existing vessels
125
role of VEGF-C in angiogenesis
main trigger of LYMPH angiogenesis
126
example of angiogenesis inhibitor?
ECM collagen
127
how do ECM collagens inhibit angiogenesis?
Endostatin/Turnstatin/Vastatin/Restin (aka NT of collagens) is cleaved when MMP cleaves collagen and inhibits angiogenesis
therefore, the product of invasion can shut down angiogenesis
128
what is the seed and soil hypothesis?
must have compatibility btwn seed (cancer cells) and soil (target organ)
some cancers tend to metastasize to very specific organs but can't really explain sites of metastasis from the pattern of circulation
129
how can we predict the site of metastais? + assay to discover this
GENE SIGNATURES can predict the site of metastasis
took tumour cells and continuously passaged thru lung/brain/bone ==> RNA seq and found that cells for lung/brain/bone each had diff gene expression
130
role of chemokines in tumour cell movement
chemokines are unique to diff organs and form gradients towards cancer cells that express chemokine receptors --> guide tumour cells towards target organ
131
6 steps of movement towards target organ w chemokines
1. tumour cells binds E-selectin
2. ECM degraded by MMP proteolysis
3. respond to chemokines
4. break barrier
5. move towards chemokines in target organ
6. begin proliferating at new organ
132
describe host-tumour interactions with adhesion
adhesion mediated by the fact that tumour cells interact with inflammatory cells and blood cells
133
4 steps of tumour cells metastasizing in liver:
1. cells transverse endothelium and enter liver
2. attach to vessel via E-selectin
3. cells enter space of Disse
4. hepatic stellate cells induce ECM deposition to create tracks for tumour cells to transverse and cause angiogenesis
134
what is a pre-metastatic niche?
organs of future metastasis are selectively and actively modified by the primary tumour even BEFORE metastatic spread has occurred --> i.e. tumour induces changes in distant microenvironment
135
what causes PMN formation?
results from combined systemic effects of tumour-secreted factors and tumour-shed extracellular vesicles
136
what INITIATES PMN formation? (3)
1. induction of leaky vessels
2. remodelling of stroma and ECM
3. systemic effects on immune system
137
describe the extracellular vesicles on cancer cells for PMN
extracellular vesicles/exosomes containing mediators will deliver the signal to target organs and determine the PMN + secrete chemokines for recruitment of cells
138
describe cancer dormancy
not every cell that enters new organ will form metastasis --> some remain dormant and may develop later
139
what process must occur once the cancer cells enter the new organ?
Mesenchymal to epithelial transition (MET)
140
Why does MET occur?
in the new organ, tumour doesn't need to be migratory anymore --> needs to be stationary
141
what occurs in MET?
re-expression glues aka collagen that hold cells together so they can expand
142
when is metabolic adaptation required?
crucial for tumour cell survival and growth in secondary organ
143
why is metabolic adaptation required?
each organ has unique metabolic landscape --> tumour cells must metabolically adapt
144
why can metabolic adaptation explain cancer dormancy
tumour cells may be unable to reprogram metabolically and stay dormant
145
why does metastasis cause a therapeutic challenge?
metastasis is dynamic --> diff stages in lifespan of metastatic cell may require diff approaches
