Richard HynesHHMI/MIT

MGH Tumor Microcirculation Course

Cambridge, MA

June 4, 2003

”Cell Adhesion in Tumor Growth, Progression and Angiogenesis"

GROWTH of PRIMARY TUMORand INITIAL INVASION

Angiogenesis-also Lymphangiogenesis (not shown)

Loss of cell adhesion

Further loss of cell adhesion

Gain of cell migration

Angiogenesis andLymphangiogenesis

• Essential for growth of primary tumor(and later of metastases)

• Involves extensive migration and adhesionof 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

U

CELL-CELL ADHESIONe.g., CADHERINS

CELL-MATRIX ADHESIONe.g., INTEGRINS

BASEMENTMEMBRANE(MATRIX)

Cell-Cell and Cell-Matrix Adhesion

Cell-cell adhesion receptors

S

S

INTEGRINS

FIBRINOGEN

S

SS

S

FIBRONECTIN LAMININ

αIIb β3 α5 β1 α6 β1

RGD

KQAGDV

CELL-MATRIX ADHESIONCell-matrix adhesion receptors

Connections between extracellularmatrix (ECM) and the actin cytoskeleton

ACTIN

ECM

PLAN VIEW

SIDE VIEW

POINTS of ATTACHMENT

The Molecular Linkage Between Actin and ECM via Integrins

β1α5

FAK TALIN

INTEGRIN

ACTINFILAMENTS

MEMBRANE

MATRIX

CDK

SIGNALLINGEVENTS

RGD

PROTEOGLYCAN

src

PKC

TENSIN

VIN

αAαA

PAXILLIN

Signals from Integrins Controlling Cell Behavior

β

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

β

Angiogenesis

Necessary for growth and survival of both

primary and metastatic tumors

αv Integrins (αvβ and αvβ5) 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:- αvβ & αvβ5integrins are proangiogenic and potential targets for antiangiogenesis therapy

Predictions from this model• Mice lacking αv integrins should show defects in angiogenesis

• So the simple predictions are not met

αv

β β

β5

βAll three areviable and fertileeither as single KOsor as double KOs

αv and β8 KOs show extensive angiogenesis, although they are not viable

β1embryonic lethal -but lacks a dozen integrins

• embryos of αv-null mice generally show normal vascular development

• the selective vascular defects in the brain are of neural/glial origin

• the βKO mouse has similar defects • in any event, they are not due to absence of

αvβand/or αvβ5 (ββ5

Conclusions from integrin knockouts

αvβand/or αvβ5 are NOT ESSENTIAL for normal vascular development

What about tumor angiogenesis?

• Transplantable tumorsHuman:

• LS180: colon carcinoma • A375SM: melanoma

Mouse:• CMT19T: lung carcinoma• B16FO: melanoma

• Endogenous tumors• RIPTAg• MMTV-neu

Tumors grown in β-null or ββ5-null mice are BIGGER than controls

B16F0

CMT19T

WT

B16F0

CMT19T

WTβ-/- ββ5-/-

Tumors grown in β-null or ββ5-null mice are BIGGER than controls

B16F0 CMT19T A375SM

p< 0.01p< 0.02 p< 0.02

Vessels in Tumors (A375M)Rag-null

Rag-null/β3-null

PECAM-1 NG-2

Tumors grown in β-null or ββ5-null mice have MORE VESSELS than controls

B16F0 tumor Normal skin

Tumor Growth and Angiogenesis

So:- tumor growth and angiogenesis are NOT dependent onαvβ orαvβ5. In fact, these integrins tend to inhibit them.

HOW?

WT βKO ββ5DKO

B16 melanoma + ++ ++

CMT19T lung carcinoma + ++ ++

LS180 adenocarcinoma + ++ ++

A375M melanoma + ++ ++

C57BL/6

Rag2

α5β1 Integrin and Fibronectin in Angiogenesis

• both are upregulated on angiogenic vessels• mice lacking α5β1 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 α5β1 integrin are proangiogenic• They appear good targets for antiangiogenesis

A new way of thinking about αv 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 αv Integrins

Transdominant Inhibition

α1β1 α2β1 α5β1

FIBRONECTIN

αvβ3 αvβ5

FIBRONECTINVITRONECTINFIBRINOGEN

OSTEOPONTINvon WILLEBRAND FACTOR

THROMBOSPONDIN

COLLAGENSLAMININS

ENDOTHELIAL CELL

SELECTIVEINHIBITION

Based on data showing cross- inhibition byligation of different integrins on the same cell.

Works best when the inhibitory integrin is at a high level

Like αvβ and αvβ5 !

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-αv integrin drugs

• It is not enough just to screen for antagonists of adhesion

• Figure out the (positive and negative) functions of αvβ and αvβ5

• 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

KERATINS VIMENTIN

FIBRONECTIN

EPITHELIAL-

MESENCHYMAL

TRANSITIONHGF/SF

Met

Common to development and tumor progression

wnt

frz

dsh

GSK3ββ

β

β

β

LEF-1

β β

-wnt

+wntAPC

TRANSCRIPTION

PDEGRADATION

IQGAP

rac42cdc

calmodulin

RELEASE of β-CATENIN from CADHERINS ENHANCES TRANSCRIPTION

How do Circulating TumorCells Arrest?

Mechanical trapping in small vessels?

Emboli with host cells and platelets?

Specific arrest via cell adhesion?

LEUKOCYTE ADHESION

PLATELET ADHESION

ROLLING ADHESION EXTRAVASATION

INVASION

CELL ADHESION in the VASCULATURE

SELECTINS β2/β1INTEGRINS,ICAMs -1VCAM

β1INTEGRINSMATRIX

β1/β2/βINTEGRINS,ICAMs -1VCAM

-1PECAM

SELECTINSINTEGRINSαIIbβetcGPIb/ /V IX,vWF ,FB ,FN CO

Could tumor cells use the same mechanisms?

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-LexS-Lex

S-LexS-Lex

PLATELETS and METASTASIS

• Platelets enhance metastatic spreadHOW?

• 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?

GPIb/V/IX

α5β1α2β1

αβ

αβ1

αIIbβ

Collagen von Willebrand factor

Fibrinogenvon Willebrand factor

FibronectinThrombospondin

Vitronectin

P-selectin

PLATELET ACTIVATION

αIIbβ

ThrombinADP

P

P

GPIb/V/IX

α5β1

α2β1

αβ

αβ1

PSGL-1

PSGL-1

PSGL-1

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex S-Lex

SELECTINS, LIGANDS, PLATELETS and METASTASIS

PLATELETS

FIBRINOGEN

ENHANCED ADHESION and TRAPPING of TUMOR CELLS ??

Chr 1

PLE

SELECTIN-DEFICIENT MICE

All strains viable and fertile

All three genes ablatedin all combinations

StephenRobinson

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

LS180 COLON CARCINOMA CELLS - Rag2-/- BACKGROUND

WT

AluPCR

SELECTIN DEPENDENCE of METASTASIS to LUNGS

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 EXPERIMENTALMETASTASIS 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 eitherhuman (LS180) or mouse (MC38)

origin

• Selectin ligands on the tumor cells may be contributing to metastasis

Endothelial CellsE E E E E E E E E E E E E

Activation

(Biosynthesis)

P P P P P P P P P P P P P PP P P

P P P

Activation

(Exocytosis)

SELECTINS on VASCULAR CELLS

L

Platelets

Leukocytes

PP

PP P

PP

PPP

PActivation

(Exocytosis)

Activation

(Shedding) L

L L

L

L

L

L

P

L

L

L

LLL

LL

L

LL

LL

L

LL L

L

L

L

L

LL

S-Lex

S-LexS-Lex

S-Lex

S-LexS-Lex

S-Lex

S-Lex

S-Lex

S-Lex S-Lex

SELECTINS, LIGANDS, PLATELETS, LEUKOCYTES and METASTASIS

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

S-Lex

P P P P P P P P P P P P P PP P P

P P P

Activation

(Exocytosis)

L L

L

L

L

L

P

P

PP

BINDING of PLATELETS to METASTATIC CELLS

Lubor BorsigTumor cells Platelets

• 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

HOST CELL ENHANCEMENTof METASTASIS

SUBCUTANEOUS INJECTION ofTUMOR 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

Pp< 0.029

Ep< 0.011

ELPp< 0.0001

33 daysTumor weight

DanielaTaverna

-/- -/- -/-

WT WT WT

DEPENDENCE on PRESENCE of L- SELECTIN

LS180 cellsRag-2-null background30 days

LuborBorsig

SELECTINS and GROWTH ofPRIMARY 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 TRANSPLANTATIONFOLLOWED by TEST for TUMOR GROWTH

Rag-2-null miceWT or

Selectin-deficient

2. Reconstitute with Bone marrow

WT or Selectin-deficient

1. Irradiate

3. After recoveryInject with tumor

cells and assayTumor growth

Enhanced tumor growth in ELP-null mice is greatly REDUCED

by irradiation and reconstitution with Rag2-/- bone marrow

Rag2BM

n=3 n=5 n=7 n=5

DanielaTaverna

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+

~10,000 genes screened -

32 are upregulated in metastases

F EXTRACELLULAR MATRIX ASSEMBLYfibronectin, collagenIα2,collagenIIIα1, ,biglycanfibromodulin

F CYTOSKELETAL ORGANIZATION,fibronectin ,RhoCthymosinβ4

α- ,cateninα- ,actininα- ,centractin -1,IQGAP calmodulin

F ANGIOGENESISfibronectin- ,t PA angiopoietin1,TGFβfamily

PLAUSIBLE CLUSTERS of ALTERED GENES

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 β4

Regulator of actin polymerizationOtherthymosins previously connected to metastasis

F RhoC

SmallGTPase- known to regulate actincytoskeleton Correlates with invasion and metastasis in human cancers

" "THREE TOP HITS

LEVEL of RhoC CONTROLS METASTASIS

A375P

A375P+ RhoC

A375M

A375M+DNRho

Cell Line # of Metastases # of Mice

A375P

A375M

A375P-RhoC

A375M-DNrho

0,0,0,0,0,1,5,10

all >100

56,70,>100, >100

13, 24, 29, 32

8

8

4

4

Pulmonary Metastases

cdc42 rac rho ROCKetc

CONTRACTILITY

IQGAP-1 CYTOSKELETALORGANIZATION

CELL SHAPE& MOTILITY

cadherinα-cateninβ-catenin CELL

ADHESION&INVASION

METASTASIS

&INTEGRINS ( )GROWTH FACTORS PDGF &INTEGRINS

( )GROWTH FACTORS LPA

-1Tiam

SMALL GTPases in INVASION and METASTASIS

Van’t Veer et al, Nature 415:530-536 (2002)

Primary breast carcinomas

Can identify an expression profile that correlates withincidence 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 distinguishesprimaries 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 whichshowed 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

17 gene signature

128 gene signature

Clusteredby all genes

17 gene signature

Kang/Massague et al Cancer Cell (in press).

Breast cancer cell line MDA-MB-231Select variants highly metastatic to boneThey “breed true”They have a characteristic expression profileTransfection of 2 or 3 of the overexpressed genes ->

increased metastasisRandom isolation and screening of clones from parent line

identifies clones with the “metastatic signature”These unselected clones ARE metastaticTherefore there ARE preexisting variant cells in the

parent populationThe “metastatic signature” is overlaid on the “poor

prognosis signature” of van’t Veer

Good prognosis Poor prognosis Metastasis

a.

Good prognosis Metastatic variants Metastasis

b.

Good prognosis Poor prognosis

c.

Metastasis

Contrasting Models for Metastatic Progression

Hynes, Cell, in press 2003

Good prognosis

Poor prognosis Metastasis

Stromal response

Metastaticvariants

A More Elaborate Model for Metastatic Progression

Hynes, Cell, in press 2003

SS

SS

SS

SS

SS

ANTIBODIES

CELL ADHESION INHIBITORS

S

S

S

S

SS

SS

SS

SS

SS

CARBOHYDRATESPEPTIDES