Richard Hynes

1
Cell Adhesion in Tumor Growth, Progression and
Angiogenesis"
Richard Hynes HHMI/MIT MGH Tumor
Microcirculation Course Cambridge, MA June 4,
2003
2
(No Transcript)
3
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
4
Angiogenesis andLymphangiogenesis

• 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

5
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

6
Cell-Cell and Cell-Matrix Adhesion
BASEMENT MEMBRANE (MATRIX)
CELL-CELL ADHESION e.g., CADHERINS
CELL-MATRIX ADHESION e.g., INTEGRINS
7
Cell-cell adhesion receptors
8
Cell-matrix adhesion receptors
9
Connections between extracellularmatrix (ECM)
and the actin cytoskeleton
PLAN VIEW
ACTIN
ECM
SIDE VIEW
POINTS of ATTACHMENT
10
The Molecular Linkage Between Actin and ECM via
Integrins
11
Signals from Integrins Controlling Cell Behavior
b
12
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?
13
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

14
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

15
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

16
Signals from Integrins Replaced by Oncogenes
b
17
Angiogenesis
Necessary for growth and survival of
both primary and metastatic tumors
18
av Integrins (av?? and av??) in Angiogenesis

• upregulated on (many) angiogenic vessels
• Inhibitors - some antibodies (LM609) and
  RGD-based peptides and peptidomimetics block
  angiogenesis and induce apoptosis in various
  model systems
• MODEL- av?? av???integrins are
  proangiogenic and potential targets for
  antiangiogenesis therapy

19
Predictions from this model

• Mice lacking av integrins should show defects in
  angiogenesis

embryonic lethal - but lacks a dozen integrins
??
??
All three are viable and fertile either as single
KOs or as double KOs
av
??
??
??
av and ?8 KOs show extensive angiogenesis,
although they are not viable

• So the simple predictions are not met

20
Conclusions from integrin knockouts

• embryos of av-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
  av???and/or av???????????? (??????????

?????av???and/or av???????????? are NOT
ESSENTIAL for normal vascular development
21
What about tumor angiogenesis?

• Transplantable tumors
• Human
• LS180 colon carcinoma
• A375SM melanoma
• Mouse
• CMT19T lung carcinoma
• B16FO melanoma
• Endogenous tumors
• RIPTAg
• MMTV-neu

22
Tumors grown in b3-null or b3/b5-null mice are
BIGGER than controls
B16F0
B16F0
WT
WT
b3-/-
?3? b?-/-
CMT19T
CMT19T
23
Tumors grown in b3-null or b3/b5-null mice are
BIGGER than controls
B16F0
CMT19T
A375SM
plt 0.02
plt 0.02
plt 0.01
24
Vessels in Tumors (A375M)
Rag-null
Rag-null/b3-null
PECAM-1
NG-2
25
Tumors grown in b3-null or b3/b5-null mice have
MORE VESSELS than controls
Normal skin
B16F0 tumor
26
Tumor Growth and Angiogenesis
WT b3KO b3/b5DKO B16
melanoma CMT19T lung
carcinoma LS180 adenocarcinoma
A375M melanoma
C57BL/6
Rag2
So- tumor growth and angiogenesis are NOT
dependent on ?vb3 or avb5. In fact, these
integrins tend to inhibit them.
HOW?
27
a5?1 Integrin and Fibronectin in Angiogenesis

• both are upregulated on angiogenic vessels
• mice lacking a5?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 a5?1 integrin are proangiogenic
• They appear good targets for antiangiogenesis

28
A new way of thinking about av 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

29
Possible Negative Regulation by av Integrins
30
Transdominant 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 avb3 and avb5?????????????????????????? !

31
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
  

32
Design of anti-av integrin drugs

• It is not enough just to screen for antagonists
  of adhesion
• Figure out the (positive and negative) functions
  of avb3 and avb?
• ?????????????for their ability to stimulate the
  negative or inhibit the positive pathways - that
  is, agonists or antagonists

33
(No Transcript)
34
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

35
EPITHELIAL- MESENCHYMAL TRANSITION
CADHERINS
HGF/SF Met
VIMENTIN
KERATINS
FIBRONECTIN
Common to development and tumor progression
36
RELEASE of b-CATENIN from CADHERINS ENHANCES
TRANSCRIPTION
37
(No Transcript)
38
How do Circulating TumorCells Arrest?
Mechanical trapping in small vessels? Emboli with
host cells and platelets? Specific arrest via
cell adhesion?
39
Could tumor cells use the same mechanisms?
40
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
41
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?

42
PLATELET ACTIVATION
?IIbb?
ADP
Thrombin
Fibrinogen von Willebrand factor
Fibronectin Thrombospondin Vitronectin
P-selectin
PSGL-1
??b?
a5b1
??b1
GPIb/V/IX
??b1
Collagen
von Willebrand factor
?IIbb?
a5b1
??b?
PSGL-1
P
??b1
??b1
PSGL-1
P
GPIb/V/IX
43
SELECTINS, LIGANDS, PLATELETS and METASTASIS
S-Lex
S-Lex
S-Lex
S-Lex
PLATELETS
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
S-Lex
FIBRINOGEN
S-Lex
S-Lex
S-Lex
ENHANCED ADHESION and TRAPPING of TUMOR CELLS ??
S-Lex
S-Lex
44
SELECTIN-DEFICIENT MICE
Chr 1
All three genes ablated in all combinations
P L E
Stephen Robinson
All strains viable and fertile
45
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

46
SELECTIN DEPENDENCE of METASTASIS to LUNGS
LS180 COLON CARCINOMA CELLS - Rag2-/- BACKGROUND
Alu PCR
WT
47
SELECTIN DEPENDENCE of METASTASIS to LUNGS
MC38 ADENOCARCINOMA CELLS -C57BL6 BACKGROUND
48
SELECTIN DEPENDENCE of METASTASIS to LUNGS
MC38 ADENOCARCINOMA CELLS - C57BL6 BACKGROUND
(GFP)
49
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

50
SELECTINS on VASCULAR CELLS
P
P
Activation (Exocytosis)
P
P
P
P
Platelets
P
P
P
P
P
P
L
Leukocytes
L
L
L
L
L
Activation (Shedding)
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
Endothelial Cells
51
SELECTINS, LIGANDS, PLATELETS, LEUKOCYTES and
METASTASIS
L
L
S-Lex
S-Lex
L
S-Lex
S-Lex
S-Lex
P
S-Lex
S-Lex
S-Lex
L
S-Lex
S-Lex
S-Lex
S-Lex
L
L
P
S-Lex
S-Lex
S-Lex
S-Lex
P
S-Lex
P
Activation (Exocytosis)
P P P P P P P P P P P P P P
P P P
P P P
52
BINDING of PLATELETS to METASTATIC CELLS
Tumor cells
Platelets
Lubor Borsig
53
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

54
SUBCUTANEOUS INJECTION of TUMOR CELLS - SCORE
GROWTH of PRIMARY TUMOR

• Mice lacking specific selectins
• Rag2 background to investigate human tumors
• (eg.LS180 adenocarcinoma)

55
Subcutaneous injection of LS180 cells into
selectin-deficient mice
33 days
Tumor weight
WT
WT
WT
-/-
-/-
-/-
Daniela Taverna
P plt 0.029
E plt 0.011
ELP plt 0.0001
56
DEPENDENCE on PRESENCE of L- SELECTIN
LS180 cells Rag-2-null background 30 days
Lubor Borsig
57
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
  ???

58
BONE MARROW TRANSPLANTATION FOLLOWED by TEST for
TUMOR GROWTH
1. Irradiate
2. Reconstitute with Bone marrow WT or
Selectin-deficient
Rag-2-null mice WT or Selectin-deficient
3. After recovery Inject with tumor cells and
assay Tumor growth
59
Enhanced tumor growth in ELP-null mice is
greatly REDUCED by irradiation and
reconstitution with Rag2-/- bone marrow
Rag2BM
Daniela Taverna
n3 n5 n7 n5
60
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

61
(No Transcript)
62
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?
63
(No Transcript)
64
(No Transcript)
65
(No Transcript)
66
(No Transcript)
67
(No Transcript)
68
LEVEL of RhoC CONTROLS METASTASIS
A375P
A375M
A375P RhoC
A375M DNRho
69
(No Transcript)
70
(No Transcript)
71
Vant Veer et al, Nature 415530-536
(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
72
(No Transcript)
73
(No Transcript)
74
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
75
(No Transcript)
76
128 gene signature
17 gene signature
Clustered by all genes
77
17 gene signature
78
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 -gt 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 vant Veer
79
Contrasting Models for Metastatic Progression
Hynes, Cell, in press 2003
80
A More Elaborate Model for Metastatic Progression
Good prognosis
Stromal response
Poor prognosis
Metastasis
Metastatic variants
Hynes, Cell, in press 2003
81
(No Transcript)