Cancer Biology 101

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Host Tumor Interactions
Ambra Pozzi, Ph.D.
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• When a tumor develops in the host (i.e. mouse
  and/or humans) it has to use the host for its
  on benefit
• growth, invasion and metastasis

Therefore the tumor can not be considered as an
independent entity. It needs constant
interactions with the host to survive
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If we dissect a tumor

• Host Cellular components
• Resident cells epithelium, fibroblasts,
  endothelial cells
• Infiltrating cells inflammatory cells, immune
  cells, endothelial cell precursors.
• Structural components
• extracellular matrix (ECM)
• Molecular components
• Cytokines, chemokines, growth factors, proteases,
  ECM fragments,
• Chemical components
• pH, 02,.

Liotta Kohn, Nature 411 375 (2001)
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Communication between tumor and host
Extracellular matrix
Inflammatory cells
Fibroblasts
Endothelial cells
Tumor Progression
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Organ environmental factors influence tumor
metastatic potential
- I.J. Fidler
Subcutaneous (in skin) encapsulated no metastases
Orthotopic injection (in colon
tissue) metastasis to regional lymph nodes and
liver
Metastatic human colon carcinoma cells
Fidler Morikawa et al, Can. Res. 48 6863, 1988
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Role of Extracellular Matrix in tumor progression
ECM components EMC-cell interaction ECM
remodeling ECM-derived cleavage products and
cell functions
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• ECM components (i.e. laminins, fibronectin,
  collagens, heparan-sulfates, vitronectin,
  nidogen,..)
• Can be part of
• 1) Stromal ECM
• 2) Basement membrane ECM

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Basement membranes are specialized structures
that separate epithelial elements from the
surrounding stroma. The two major
representatives of the components of basement
membranes are non-fibrillar collagens and
laminins.
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Basement membranes
For about two decades it has been apparent that
all BMs contain traditional ECM components i.e.
laminins, Type IV collagen, and
proteoglycans. Recently this complexity has
increased as new components are described The
entactin/nidogen family and the new Heparan
Sulfate Proteoglycans Agrin and Type XVIII
collagen
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The three steps of invasion
1
2
Matrix attachment
Matrix degradation
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Locomotion
Liotta, LA. Tumor invasion and metastasis-role of
the extracellular matrix. Cancer Res 46 (1986)
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Collagen IV
Collagen IV is a non fibrillar collagen virtually
found in all the BMs of the body
Collagen IV is composed of six different chains
called a1-a6 (IV) chains.
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collagenous
NC
a1 assembles with a2 most represented form of
collagen IV a3 assembles with a4 and a5 found
in the kidneys (GBM) in lung (perivascular),
testis, and eyes a5 assembles with a6 found
primarily in skin, smooth muscle, esophagus, and
kidney (Bowman's capsule).
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Collagen XVIII (HSPG)
Collagen XVIII is a non fibrillar collagen
composed by one chain a1(XVIII)
Collagen XVIII is abundant in the retina,
epidermis, cardiac and striated muscle, kidney,
blood vessels, and lung.


Collagen XVIII, is the second most abundant BM
collagen next to collagen IV
Several Ser-Gly dipeptides suitable for GAG
attachment are found in the collagen XVIII
sequence, raising the suspicion that collagen
XVIII was a PG.
Glycosaminoglycans (GAGs) represent the sulfated
carbohydrate of proteoglycans
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Collagen XVIII formed by the assembly of tree
alpha1 chains
B
11 NC domains
a1a1a1
a1
-COOH
NH2-
10 collagenous domains
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Laminins

• Primarily BM components formed by the assemby of
  three chains, alpha, beta and gamma chains.
• Belong to a multigenic family and the most
• Studied are Ln1, Ln5 and Ln10/11

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Laminin 5
COMPOSED OF THREE CHAINS (a3b3g2) WIDELY
EXPERESSED IN ALL BMs
globular domain
-COOH
b3
g2
NH2-
-NH2
a3
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The ECM undergoes constant remodeling and
degradation under physiological (wound healing,
embryonic development, menstruation and tissue
remodeling) as well as pathological (arthitis,
tumor progression) conditions
degradation
Neo-synthesis
Proteolytic enzymes Cathepsins, uPA/Plasmin, MMPs
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• ECM-derived cleavage products and cell functions

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Which cell functions can be regulated by ECM
products?? Angiogenesis
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When does angiogenesis occur?
The development of new blood vessels from
preexisting vessels

• During physiological events
• Wound healing
• Menstruation
• Pregnancy
• Embryo formation
• Tissue remodeling
• During pathological events
• Tumor growth and development

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• The major steps involved in angiogenesis are
• EC proliferation
• EC migration
• Tube formation
• Blood flow

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What is the link between proteases/BM
components/angiogenesis
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What is the role of Collagen XVIII
B
11 NC domains
a1
-COOH
NH2-
10 collagenous domains
A 28 kDa N-terminal cleavage of collagen XVIII
has been identified as potent inhibitor of EC
functions and called ENDOSTATIN
Matrilysin (MMP7), Gelatinase A (MMP2), Cathepsin
L and pancreatic elastase can cleave collagen
type XVIII NC1 domain and generation of a 28-kDa
fragment.
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How does Endostatin exert its action????
Wickstrom et al An endostatin-derived peptide
interacts with integrins (b1-containing) And
regulates actin cytoskeleton and migration of
endothelial cells.J Biol Chem. 2004
Rehn et al Interaction of endostatin with
integrins (a5 and av) implicated in
angiogenesis.PNAS 2001
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• Eriksson et al.
• Endostatin can compete with b-FGF and VEGF for
  heparan
• sulfate chain biding thus preventing the storage
  of this potent
• angiogenic molecules in the ECM milieu

Lee et al Endostatin binds to the catalytic
domain of MMP2 preventing its action. FEBS
Lett. 2002
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What is the role of collagen IV
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Recombinant Human a(IV) NC1 Domains Inhibit
Angiogenesis In Vivo
bFGF a2 NC1
bFGF a1 NC1
Not Treated
bFGF
bFGF a4 NC1
bFGF a5 NC1
bFGF a3 NC1
bFGF a6 NC1
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• a1 arresten integrin a1b1
• a2 angiocol integrin a1b1
• a3 tumstatin integrin avb3 and less extent
  avb3
• a4 not tested
• a5 not tested
• a6 not tested

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Which cell functions can be regulated by ECM
products?? Tumor cell growth
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The collagenous domain of collagen IV

• collagen IV alters cell functions via its triple
  helix domain/s

melanoma cells, for example, adhere, spread and
become more motile in response to a triple
helix-rich domain, while no effects are observed
after stimulation with the NC1 domains.
Three triple helix-rich domains within the a1(IV)
chain, namely the CB3, the 531-543 and 1263-1277
regions, have been shown to affect the functions
of different tumor types.
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What is the role of Laminin 5?
globular domain
-COOH
b3
g2
NH2-
-NH2
a3
Adhesion Ln-5 binds to the a6b1, a6b4 and a3b1
integrins, presumably through its G domain and
the a6b4 integrin seems to be the most critical
for epidermal-dermal stability
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a3
A
Plasmin
Alteration of cell functions (the role of alpha
chain) The a3 chain cleavage of Ln-5 by plasmin
from 190 kDa to 160 kDa leads to decreased cell
motility
COOH
NH2
160 kDa anti-migratory
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b3
B
Alteration of cell functions (the role of beta
chain) MT1-MMP cleaves the purified b3 chain of
human Ln-5 to an 80-kDa fragment and this
cleavage process enhances cell migration.
COOH
80kDa pro-migratory
MT1-MMP
?
NH2 -
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Alteration of cell functions (the role of gamma
chain) Robust cell migration on Ln-5 is initiated
after cleavage of its g2-chain by MMP-2 and
MT1-MMP Domain III (EGF-like repeats) binds to
the EGF receptor promoting cell migration.
g2
C
COOH
MMP2, MT1-MMP
-NH2
g2 pro-migratory
DIII activation of erbB1
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In conclusion

• ECM components are not static.
• BM components (proteins, HSPG) can modulate cell
  functions as whole or as
• proteolytic fragments
• Proteases by cleaving these ECM play a relevant
  role in modulating EC function

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Role of cytokine/receptors/proteolytic enzymes
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• Chemokines
• - represent a family of small-molecular-weight (8
   14 kDa) chemotactic molecules
• that bind to G-protein-coupled receptors.
• first chemokine to be identified was IL-8. Other
  chemokines include platelet factor 4, platelet
  basic protein, melanoma growth stimulatory
  protein, macrophage inflammatory protein 2, etc.
• CXC(L) and CXC(R) is the new nomenclature
• been thought to be primarily as leukocyte
  attractants
• contribute to a number of tumor-related
  processes, such as tumor cell growth,
  angiogenesis/angiostasis, local invasion, and
  metastasis.

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Can the host guide tumor cells by expressing
particular receptors/ligands?

• CXCR4 receptor on breast cancer cells
• CXCL12 ligand in lung, liver, bone
• Ligand/receptor interaction cell migration
  (invasion and metastasis)
• antibody blocks lung mets in animal models
• Some drugs in development

Muller et al. Nature 410 50-56 2001
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Role of Endothelial cells in tumor progression
homing receptors
Zhu DZ et al. Mediation of lung metastasis of
murine melanomas by a lung-specific endothelial
cell adhesion molecule. PNAS 88 9568-9572
(1991). Not all the endothelial cells in the
body are the same..some of them express homing
receptors for tumor cells
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Role of Receptors
ab
Integrin a6b4 helps the epithelial cells to bind
to the basement membrane. Down-regulation of
a6b4 leads to decreased attachment to the
basement membrane and increased
migration/invasion. Integrin a6b4
beneficial to the host as it prevents epithelial
cell
migration/invasion
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Role of fibroblasts
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Fibroblasts influence epithelial cell tumor
progression
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Fibroblasts make epithelial cells tumorigenic
10
20 - 5000
Cunha Olumi et al, Can. Res. 59 5002, 1999
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Inflammatory and Immune cells influence tumor
progression
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The good immune system
Immune surveillance
Natural Killer (NK) cell
Immunity 21, 137, 2004
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The good immune system
Cytokines are small molecules that are the core
of communication between immune system
cells, and/or immune system cells and other
tissue types. They are actively secreted by
immune cells. Their action is often local Some
of the better known cytokines include histamine,
prostaglandin, TNF-a, IFNs IL-1, and IL-2 and so
on Cytokines act by binding to their
cell-specific plasma membrane receptors and
then promote migration/proliferation/adhesion.
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Chemokines and cytokines have both pro- and
anti-tumorigenic effects
Cousssens Werb Nature 420 860 (2002)
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The same can be said for matrix
metalloproteinases (MMPs)
MMPs 1) proteolytic enzymes involved in the
degradation of extracellular matrix components
2) they can be produced/secreted by both host and
tumor cells
- MMPs (i.e. MMP-null mice) no or delayed tumor
growth/progression
MMPs (tumor cells) increased tumor growth and
migration/invasion
MMPs (host/tumor cells) increased
production of extracellular matrix fragments able
to prevent both vessel and tumor cell growth.
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Role of platelets in tumor metastasis

• Platelets beneficial effect to the host as they
  participate in the clot formation and coagulation
  thus avoiding bleeding
• Platelets beneficial to the tumors.
• Platelets can promote tumor metastasis by
• Being a chaperone for tumor cells
• Secreting angiogenic factors (i.e. VEGF)

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In conclusion
Tumors can not considered as independent
entities. In fact, they need constant
interaction with the host

• The host components contributing to tumor
  growth/progression can be
• Cellular (i.e. fibroblasts and endothelial cells)
• Structural (i.e. ECM)
• Molecular (cytokines, MMPs, receptors)
• Chemical (pH, O2)

Studying the host is important as 1) Host cells
unlike tumor cells are less susceptible to
genetic instability 2) Might offer an excellent
therapeutic target
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Why are we interested in the stroma? lack of
genetic instability in stromal cells
Genetic instability Drug resistance
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Lethal tumor burden
TUMOR PROGRESSION
Malignant
Benign
TIME
Natural lifespan
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How can we study the influence of the host in
tumor development?

• If we use animal models (i.e. mouse models) we
  can manipulate the
• Host (mouse)

i.e. Protein X is expressed only by endothelial
cells and it has been shown to accelerate tumor
angiogenesis and increase blood supply.
Addressing the problem Generate mice lacking
protein X only in endothelial cells (conditional
KO) and determine the ability of tumor cells to
grow in these mice
Pitfall Protein X is essential for endothelial
cell survival, without that protein The mouse
wont develop
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• We can think of manipulating tumor cells only

i.e. Protein Y is expressed by both fibroblasts
(host) and by tumor cells and it has been found
up-regulated in both tumors and stromal
components in malignant melanomas.
Addressing the problem We could create in vitro
tumor cells (i.e. melanoma) lacking protein Y and
inject these cells into mice. This experiment
might tell us whether stromal-derived Protein Y
is per se sufficient to promote malignancy.
Pitfall Protein Y is essential for some specific
tumor cell functions, without that protein tumor
cells might not migrate/invade.
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Can we visualize tumor cells in the host?
Tumor cells can be tagged With fluorescent
markers i.e. GFP-tumor cells
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How can we target the host to prevent tumor
grow/spreading?
Endothelial cells form vessels for blood supply
and they are somehow easy and/or ideal
target So tumor-bearing mice (or humans) can be
treated with drug, molecules directly targeted
to endothelial cells to try to block pathological
blood supply.