Transcript Angiogenesis

”Cell Adhesion in Tumor Growth,
Progression and Angiogenesis"
Richard Hynes
HHMI/MIT
MGH Tumor Microcirculation Course
Cambridge, MA
June 4, 2003
GROWTH of PRIMARY TUMOR
and INITIAL INVASION
Loss of cell adhesion
Further loss of
cell adhesion
Angiogenesis
-also Lymphangiogenesis
(not shown)
Gain of cell migration
Angiogenesis and
Lymphangiogenesis
• Essential for growth of primary tumor
(and later of metastases)
• Involves extensive migration and adhesion
of endothelial cells and pericytes
• Involves organization of basement membranes
Metastatic Spread
• Intravasation
• Survival in circulation
• Arrest at a distant site - selectivity??
• Intravascular Proliferation ?
• Extravasation
• Survival and proliferation at the new site
• Angiogenesis again
• All of these involve cell adhesion
Cell-Cell and Cell-Matrix Adhesion
U
CELL-CELL ADHESION
e.g., CADHERINS
BASEMENT
MEMBRANE
(MATRIX)
CELL-MATRIX ADHESION
e.g., INTEGRINS
Cell-cell adhesion receptors
Cell-matrix
adhesion
receptors
CELL-MATRIX
ADHESION
INTEGRINS
KQAGDV
S
S
S
S
IIb 
S
S
5 
6 
RGD
FIBRINOGEN
FIBRONECTIN
LAMININ
Connections between extracellular
matrix (ECM) and the actin cytoskeleton
PLAN VIEW
ACTIN
ECM
SIDE VIEW
POINTS of ATTACHMENT
The Molecular Linkage Between
Actin and ECM via Integrins
SIGNALLING
EVENTS
ACTIN
FILAMENTS
TALI
N
PAXILLIN
IN
S
N
E
T
VIN
FAK
A
src
A
PKC
CDK
MEMBRANE
INTEGRIN

MATRIX

RGD
PROTEOGLYCAN
Signals from Integrins
Controlling Cell Behavior

Functions of Cell Adhesion Receptors
• Mediate adhesion to adjacent cells and to ECM
• Control cell shape, polarity and migration
• Control cell proliferation, survival,
gene expression and differentiation
How do these functions impact tumor progression?
MATRIX/INTEGRINS and GROWTH CONTROL
• Integrins regulate cyclin D synthesis
• Integrins regulate PIP2 synthesis
• Both these effects synergize with stimulation by
soluble growth factors
• In fact, they are necessary for growth factors to
promote growth - cells will not grow with
growth factors alone - they need matrix
attachment through integrins.
• This is “anchorage dependence of growth”
MATRIX/INTEGRINS and CELL SURVIVAL
• Integrins regulate PI3 kinase and Akt,
acting through FAK
• This pathway suppresses apoptosis
• So extracellular matrix, acting via integrins
provides local survival signals
• i.e., cells must be attached to the correct matrix
in order to survive.
• This is “anchorage dependence of survival”
ANCHORAGE DEPENDENCE
• Most normal cells are dependent on anchorage
for survival and proliferation
• Tumor cells are not, because oncogenes provide
the signals normally provided by
integrins and other adhesion receptors
• So tumor cells are less dependent on being
attached in the correct place
Signals from Integrins
Replaced by Oncogenes

Angiogenesis
Necessary for growth and survival of both
primary and metastatic tumors
v Integrins (v and v)
in Angiogenesis
• upregulated on (many) angiogenic vessels
• Inhibitors - some antibodies (LM609) and RGDbased peptides and peptidomimetics block
angiogenesis and induce apoptosis in various
model systems
• MODEL:- v & vintegrins are
proangiogenic and potential targets
for antiangiogenesis therapy
Predictions from this model
• Mice lacking v integrins should show defects in angiogenesis
embryonic lethal but lacks a

dozen integrins

v



All three are
viable and fertile
either as single KOs
or as double KOs
v and 8 KOs show extensive angiogenesis,
although they are not viable
• So the simple predictions are not met
Conclusions from integrin knockouts
• embryos of v-null mice generally show
normal vascular development
• the selective vascular defects in the brain
are of neural/glial origin
• the KO mouse has similar defects
• in any event, they are not due to absence of
vand/or v
(

vand/or v
are NO
ESSENTIAL for normal vascular development
What about tumor angiogenesis?
• Transplantable tumors
Human:
• LS180: colon carcinoma
• A375SM: melanoma
Mouse:
• CMT19T: lung carcinoma
• B16FO: melanoma
• Endogenous tumors
• RIPTAg
• MMTV-neu
Tumors grown in -null or -null
mice are BIGGER than controls
B16F0
B16F0
-/-
WT
CMT19T
 -/-
WT
CMT19T
Tumors grown in -null or -null
mice are BIGGER than controls
B16F0
p< 0.02
CMT19T
p< 0.01
A375SM
p< 0.02
Vessels in Tumors (A375M)
Rag-null
Rag-null/3-null
PECAM-1
NG-2
Tumors grown in -null or -null
mice have MORE VESSELS than controls
B16F0 tumor
Normal skin
Tumor Growth and Angiogenesis
WT
KO
DKO
B16 melanoma
+
++
++
CMT19T lung carcinoma
+
++
++
LS180 adenocarcinoma
+
++
++
A375M melanoma
+
++
++
C57BL/6
Rag2
So:- tumor growth and angiogenesis are NOT dependent on
v or v. In fact, these integrins tend to inhibit them.
HOW?
51 Integrin and Fibronectin in
Angiogenesis
• both are upregulated on angiogenic vessels
• mice lacking 51 die with vascular defects
• mice lacking
die with vascular
defects
• antibodies to either inhibit angiogenesis
• peptides blocking their interaction inhibit
angiogenesis
• that is - genetics and inhibitor studies conform here
• Fibronectin and 51 integrin are proangiogenic
• They appear good targets for antiangiogenesis
A new way of thinking about v
integrins in angiogenesis
• The original model of their being proangiogenic
does not explain all the data
• Perhaps they are actually antiangiogenic or
negative regulators some or all the time
• The negative regulation model does a better,
although not a perfect job of explaining the data
Possible Negative Regulation by v Integrins
Transdominant Inhibition
ENDOTHELIAL CELL
11
21
COLLAGENS
LAMININS
v3 v5
FIBRONECTIN
VITRONECTIN
FIBRINOGEN
OSTEOPONTIN
von WILLEBRAND FACTOR
THROMBOSPONDIN
51
FIBRONECTIN
SELECTIVE
INHIBITION
Based on data showing cross- inhibition by
ligation of different integrins on the same cell.
Works best when the inhibitory integrin is at a high level
Like v and
v
!
Agonists orAntagonists?
• That often depends on the assay
• The same agent can act as an agonist when
presented on a substrate and an antagonist
when presented in solution
• An agent detected as an antagonist in an
adhesion assay can be an agonist with
respect to signaling
Design of anti-v integrin drugs
• It is not enough just to screen for
antagonists of adhesion
• Figure out the (positive and negative)
functions of v and v
•
for their ability to
stimulate the negative or inhibit the positive
pathways - that is, agonists or antagonists
LOSS of ADHESION for HOME BASE
LOSS of CELL-CELL ADHESION
PROTEINS (CADHERINS)
CONTRIBUTES to INVASION
of COLON and STOMACH CANCERS
GAIN of NEW ADHESION
GAIN of NEW CELL ADHESION
PROTEINS (INTEGRINS )
CONTRIBUTES to INVASION
of MALIGNANT MELANOMA
Cadherins and Integrins in
Tumor Invasion
• Cadherins, particularly E-cadherin, are frequently
lost from invasive malignant tumors
• Integrins are sometimes gained by invasive tumors
• This reflects the switch from sessile adherent epithelial
cells to migratory, invasive mesenchymal cells
• Often called the Epithelial-Mesenchymal Transition
or EMT
CADHERINS
EPITHELIALMESENCHYMAL
TRANSITION
HGF/SF
Met
KERATINS
VIMENTIN
FIBRONECTIN
Common to development and tumor progression
RELEASE of -CATENIN from CADHERINS
ENHANCES TRANSCRIPTION
wnt
frz
dsh


P
GSK3

APC
DEGRADATION
- wnt
+ wnt



LEF-1
TRANSCRIPTION
rac
cdc42
IQGAP
calmodulin
How do Circulating Tumor
Cells Arrest?
Mechanical trapping in small vessels?
Emboli with host cells and platelets?
Specific arrest via cell adhesion?
CELL ADHESION in the VASCULATURE
SELECTINS
INTEGRINS
IIb3 etc
GPIb/V/IX
vWF, FB, FN, CO
PLATELET ADHESION
LEUKOCYTE ADHESION
ROLLING
SELECTINS
ADHESION
2/1 INTEGRINS
ICAMs, VCAM-1
EXTRAVASATION
1/2 / INTEGRINS
ICAMs, VCAM-1
PECAM-1
INVASION
Could tumor cells use the same mechanisms?
1 INTEGRINS
MATRIX
SELECTINS and METASTASIS
• Acquisition by human carcinomas of
carbohydrate ligands (S-Lex and S-Lea) for
selectins is associated with poor prognoses
• Selectins are expressed by vascular cells platelets, leukocytes, endothelium
• Could tumor cells use selectins in their
metastatic spread?
S-Lex
S-Lex
S-Lex
S-Lex
PLATELETS and METASTASIS
• Platelets enhance metastatic spread
•
HOW?
Provision of adhesion molecules
• Adherence to tumor cells?
• Bridging between tumor cells and endothelium ?
• Provision of growth factors/cytokines
• Protection against turbulence
• Trapping of embolus
• Could selectins or integrins play a role?
PLATELET ACTIVATION
ADP
PSGL-1
IIb
P-selectin

Thrombin
 


GPIb/V/IX
Collagen von Willebrand factor
Fibrinogen
von Willebrand factor
Fibronectin
Thrombospondin
Vitronectin
IIb

PSGL-1
 
P


P
GPIb/V/IX
PSGL-1
SELECTINS, LIGANDS, PLATELETS and METASTASIS
S-Lex
S-Lex
S-Lex
PLATELETS
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
FIBRINOGEN
S-Lex
S-Lex
ENHANCED ADHESION and
TRAPPING of TUMOR CELLS ??
SELECTIN-DEFICIENT MICE
Chr 1
All three genes ablated
in all combinations
P
L
E
Stephen
Robinson
All strains viable and fertile
INTRAVENOUS INJECTION of TUMOR
CELLS - SCORE LUNG METASTASES
• Mice lacking one, two or all three selectins
• C57BL6 background to investigate murine tumors
(eg.,MC38 colon adenocarcinoma)
• Rag2-/- background to investigate human tumors
(eg.LS180 adenocarcinoma)
• These cells express ligands for all 3 selectins
Daniela Taverna and collaboration with Ajit Varki/Lubor Borsig
SELECTIN DEPENDENCE of METASTASIS to LUNGS
LS180 COLON CARCINOMA CELLS - Rag2-/- BACKGROUND
Alu
PCR
WT
SELECTIN DEPENDENCE of METASTASIS to LUNGS
MC38 ADENOCARCINOMA CELLS -C57BL6 BACKGROUND
SELECTIN DEPENDENCE of METASTASIS to LUNGS
MC38 ADENOCARCINOMA CELLS - C57BL6 BACKGROUND
(GFP)
SELECTINS and EXPERIMENTAL
METASTASIS to LUNGS
• P and L selectins both enhance metastasis and
their effects are additive
• E-selectin has rather little effect
• True for injected tumor cells of either
human (LS180) or mouse (MC38) origin
• Selectin ligands on the tumor cells may be
contributing to metastasis
SELECTINS on VASCULAR CELLS
Platelets
Leukocytes
L
L
Activation
P
P
P
L
L
L
L
L
L
L
L
PPP
Activation
L
(Shedding)
L
L
PPP
L
P
L
L
L
L
L
L
Activation
P
P
P P
L
L
L
L
L
P
P
(Exocytosis)
L
L
P
L
L
L
L
PP PPPPPPPPPPPP
(Exocytosis)
Endothelial Cells
Activation
(Biosynthesis)
EEEEEEEEEEEEE
SELECTINS, LIGANDS, PLATELETS,
LEUKOCYTES and METASTASIS
L
S-Lex
L
S-Lex
S-Lex
P S-Lex
S-Lex
P
S-Lex
S-Lex
S-Lex
S-Lex
P
PPP
L
S-Lex
S-Lex
x
S-Lex S-Le
L
P
Activation
PPP
L
S-Lex
S-Lex
S-Lex
S-Lex
(Exocytosis)
PP PPPPPPPPPPPP
L
BINDING of PLATELETS
to METASTATIC CELLS
Tumor cells
Platelets
Lubor Borsig
HOST CELL ENHANCEMENT
of METASTASIS
• Likely contributors include platelets and leukocytes
binding to the tumor cells
• Suggests that reagents blocking selectin interactions
might be useful in inhibiting metastatic spread
• Need to find out which are the key host cells
e.g, bone marrow transplantations
SUBCUTANEOUS INJECTION of
TUMOR CELLS - SCORE
GROWTH of PRIMARY TUMOR
• Mice lacking specific selectins
• Rag2 background to investigate human tumors
(eg.LS180 adenocarcinoma)
Subcutaneous injection of LS180 cells into selectin-deficient mice
Tumor weight
WT
P -/p< 0.029
33 days
WT
E -/p< 0.011
WT
ELP-/p< 0.0001
Daniela
Taverna
DEPENDENCE on PRESENCE of L- SELECTIN
LS180 cells
Rag-2-null
background
30 days
Lubor
Borsig
SELECTINS and GROWTH of
PRIMARY TUMORS
• Deficiencies in P, L and E-selectins all enhance
tumor growth and the effects are additive
• True for several different tumor cell lines
• Suggests some anti-tumor role for leukocytes
• Rag-2 -/- mice lack B, T and NK-T cells
• Macrophages, NK cells, platelets, endothelium ???
BONE MARROW TRANSPLANTATION
FOLLOWED by TEST for TUMOR GROWTH
1. Irradiate
Rag-2-null mice
WT or
Selectin-deficient
2. Reconstitute with
Bone marrow
WT or
Selectin-deficient
3. After recovery
Inject with tumor
cells and assay
Tumor growth
Enhanced tumor growth in ELP-null mice is greatly REDUCED
by irradiation and reconstitution with Rag2-/- bone marrow
Daniela
Taverna
Rag2BM
n=3 n=5 n=7 n=5
CONCLUSIONS from
BONE MARROW TRANSPLANTS
• Mice with selectin-deficient bone marrows
consistently yield larger tumors
• Some selectin-dependent BM-derived cells
suppress tumor growth
• Macrophages and NK cells express L-selectin and PSGL-1
• Endothelium expresses P- and E-selectins
• Platelets express P-selectin and PSGL-1 platelets could also recruit other cell types
How do metastatic cells arise?
Are they all the same?
Is there specificity in their arrest?
Or is there specificity in their ability to
grow/survive in distant sites?
Cell Line
Cells Injected
Tumors in Lungs
A375P
5x105
0,0,0,0,5
A375M1
1x105
all 50+
A375M2
2x105
~50
B16F0
5x104
0,0,0,3,5
B16F1
5x104
50+ to solid
B16F2
2.5x104
all 50+
PLAUSIBLE CLUSTERS of ALTERED GENES
~10,000 genes screened 32 are upregulated in metastases
F EXTRACELLULAR MATRIX ASSEMBLY
fibronectin, collagenI2,
collagenIII1, biglycan, fibromodulin
F
CYTOSKELETAL ORGANIZATION
fibronectin, RhoC, thymosin 4
-catenin, -actinin, -centractin,
IQGAP-1, calmodulin
F ANGIOGENESIS
fibronectin
t-PA, angiopoietin 1, TGF family
THREE "TOP" HITS
F
FIBRONECTIN
Extracellular matrix protein.
Known to promote cell proliferation and cell survival
Known to promote cell migration
Known to promote angiogenesis
Upregulated in some other metastatic cells
F
THYMOSIN 4
Regulator of actin polymerization
Other thymosins previously connected to metastasis
F
RhoC
Small GTPase - known to regulate actin cytoskeleton
Correlates with invasion and metastasis in human cancers
LEVEL of RhoC CONTROLS
METASTASIS
A375P
A375P
+ RhoC
A375M
A375M
+DNRho
Pulmonary Met ast ases
Cell Line
# of Metastases
# of Mice
A3 7 5P
0 ,0 ,0 ,0 ,0 ,1 ,5 ,1 0
8
A3 7 5 M
all >1 0 0
8
A3 7 5 P-RhoC
5 6 ,7 0 ,>1 0 0 , >1 0 0
4
A3 7 5 M-DNrho
1 3, 24, 29, 32
4
SMALL GTPases in INVASION and METASTASIS
INTEGRINS &
GROWTH FACTORS (PDGF)
INTEGRINS &
GROWTH FACTORS (LPA)
Tiam-1
cdc42
rac
IQGAP-1
cadherin
-catenin -catenin
rho
CYTOSKELETAL
ORGANIZATION
CELL
ADHESION
ROCK
etc
CONTRACTILITY
CELL SHAPE
& MOTILITY
INVASION &
METASTASIS
Van’t Veer et al, Nature 415:530-536 (2002)
Primary breast carcinomas
Can identify an expression profile that correlates with
incidence of metastases
Suggests bulk primary tumor already has properties that
predispose to metastasis
That is, not (only) rare variant metastatic cells
Ramaswamy et al, Nature Genetics 33: 49-54 (2003)
Miscellaneous collection of 12 metastases and 64 primary tumors
of same histological types - all adenocarcinomas
Can identify an expression profile of 128 genes that distinguishes
primaries from metastases
Some primaries show the “metastasis pattern”
Analyzed available data sets and found that the 128 gene set
could split primaries into two sets, one of which
showed the “metastasis pattern” and had poor prognosis
- same result with a 17 gene set
Suggests bulk primary tumors already have properties that
predispose to metastasis
That is, not (only) rare variant metastatic cells
128 gene
signature
17 gene
signature
Clustered
by all genes
17 gene signature
Kang/Massague et al Cancer Cell (in press).
Breast cancer cell line MDA-MB-231
Select variants highly metastatic to bone
They “breed true”
They have a characteristic expression profile
Transfection of 2 or 3 of the overexpressed genes ->
increased metastasis
Random isolation and screening of clones from parent line
identifies clones with the “metastatic signature”
These unselected clones ARE metastatic
Therefore there ARE preexisting variant cells in the
parent population
The “metastatic signature” is overlaid on the “poor
prognosis signature” of van’t Veer
Contrasting Models for Metastatic Progression
a.
Good prognosis
Poor prognosis
Metastasis
Metastatic variants
Metastasis
b.
Good prognosis
c.
Good prognosis
Poor prognosis
Metastasis
Hynes, Cell, in press 2003
A More Elaborate Model for
Metastatic Progression
Good prognosis
Stromal
response
Metastatic
variants
Poor prognosis
Metastasis
Hynes, Cell, in press 2003
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CELL ADHESION INHIBITORS
SS
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CARBOHYDRATES
PEPTIDES
ANTIBODIES
S
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