Yanlin Yu, Ph.D.National Cancer Institute, National Institutes of Health

Bethesda, Maryland

Mechanisms of Metastasis Regulation

What the Mouse Can Tell Us

Mechanism

Molecular/GeneticPathways

Discovery

MolecularTargets

Preclinical Studies

Therapy, Prevention &Risk Assessment

Integration of Basic and Translational Approaches in Mouse Cancer Models

Human

Human Comparisons Help:Validate Mouse Model

Prioritize Research Efforts Human

Human

Mechanisms in Progression to the Metastatic State in Rhabdomyosarcoma

Ezrin and Six1 as key metastatic regulatorsWhat is the relationship between Ezrin and Six1?Histone modifications at the ezrin locus are linked to Ezrin expression

Pathway Assessment of Metastasis in melanoma

Constitutive c-Met signaling through a nonautocrine mechanism

promotes metastasis Mechanisms of PTEN-mediated metastatic suppression

Mechanisms in Progression to the Metastatic State in Rhabdomyosarcoma

Ezrin and Six1 as key metastatic regulators

Rhabdomyosarcoma Rhabdomyosarcoma (RMS)(RMS)

• Thought to arise from skeletal muscle Thought to arise from skeletal muscle precursors (satellite cells or myoblasts)precursors (satellite cells or myoblasts)

• Approximately 250 newly diagnosed Approximately 250 newly diagnosed cases a year in the U.S.cases a year in the U.S.

• Most common soft-tissue sarcoma of Most common soft-tissue sarcoma of childhoodchildhood

• Patients diagnosed with advanced Patients diagnosed with advanced disease have a dismal prognosisdisease have a dismal prognosis

RMS Molecular PathogenesisRMS Molecular Pathogenesis

• Hallmarks of Alveolar RMS:Hallmarks of Alveolar RMS:

– t(2;13)(q35;q14) fuses t(2;13)(q35;q14) fuses Pax3Pax3 with with FKHR FKHR oror

– Variant t(1;13) fuses Variant t(1;13) fuses Pax7Pax7 with with FKHRFKHR

• Ubiquitous overexpression of IGF2Ubiquitous overexpression of IGF2

• Other reported alterations:Other reported alterations:

– N-N-MYCMYC (amplification) (amplification)

– N-N-RASRAS/K-/K-RASRAS (point mutation) (point mutation)

– p53p53 pathway ( pathway (p53p53 mutation; mutation; MDM2MDM2 amplification) amplification)

– pRBpRB pathway ( pathway (p16INK4ap16INK4a deletion; deletion; CDK4CDK4 amplification) amplification)

– Overexpression of c-MET (HGF/SF receptor)Overexpression of c-MET (HGF/SF receptor)

Original Goal:Original Goal:

To study the genetic interactions between c-To study the genetic interactions between c-MET signaling and the MET signaling and the INK4a/ARFINK4a/ARF locus locus

HGF/SF-Met Signal TransductionHGF/SF-Met Signal Transduction

HGF/SF-Met Induces:

ProliferationMigration/Scattering

MorphogenesisInvasiveness

SurvivalAngiogenesis

Genetic Analysis of the Effect of ink4a/arfloss in HGF/SF transgenic mice

Selective induction of rhabdomyosarcoma inSelective induction of rhabdomyosarcoma inInk4a/Arf-deficient HGF/SF-transgenic miceInk4a/Arf-deficient HGF/SF-transgenic mice

HGF/SFink4a/arf+/-

HGF/SFink4a/arf-/-

ink4a/arf+/+HGF/SF

ink4a/arf-/-

HGF/SFink4a/arf+/-

HGF/SFink4a/arf-/-

ink4a/arf+/+HGF/SF

ink4a/arf-/-

Molecular Analysis of Primary RMS TissueMolecular Analysis of Primary RMS Tissue

Mouse RMS: RNA

Mouse RMS: DNA

ink4a/arf

p-MET

MET

Human RMS: Protein

Richard Sharp & Yanlin Yu in Nature Medicine, 2002

Disruption of c-MET andDisruption of c-MET andINK4a/ARF Pathways inINK4a/ARF Pathways inHuman and Mouse RMSHuman and Mouse RMS

PreclinicalStudies

Molecular & FunctionalValidation of Candidates

Establish StableMetastatic Cell Lines

Global Search forNovel Targets

Analysis of Tumor Progression in Genetically EngineeredMouse Models of Human Cancer

• Establish RMS cell lines and characterize their metastatic potential

• Perform array-based expression profiling on this panel of cell lines

• Identify and validate differential expression of candidate genes

•Perform functional studies to test hypotheses about candidate genes

Expression Profiling of Metastasis in Rhabdomyosarcoma

STRATEGY

RMS Cell Lines are Either Highly or Poorly Metastatic

Cell Line No. Tail Vein Injection Orthotopic Transplant

RMS2 Extensive ExtensiveRMS7 ExtensiveRMS10 Extensive ExtensiveRMS11 Extensive ExtensiveRMS14 Moderate ExtensiveRMS29 ExtensiveRMS31 Extensive ExtensiveRMS33 ExtensiveRMS54 Extensive

RMS13 None LightRMS32 None LightRMS43 None ExtensiveRMS114 None RMS117 None RMS119 None RMS122 None RMS123 NoneRMS740 None LightRMS772 None None

Gross Pulmonary Metastasis

Dendrogram from Cluster Analysis of Highly vs.

Poorly Metastatic rhabdomyosarcoma (RMS) Cell Lines

Identification of Novel Metastasis GenesExpression profiling of RMS cell lines

Yu, et al. Nature Medicine, 2004

overexpressed

underexpressed

Highly Metastatic Poorly Metastatic

•Homeoprotein transcription factor•Required for development of skeletal muscle•Elevated in human breast cancer

Six1 (sine oculis-related homeobox 1)

Ezrin (Villin 2)•Links plasma membrane to cytoskeleton•Resides at nexus of “metastasis” pathways (survival, motility, invasiveness, adherence)•Implicated in osteosarcoma & adenocarcinoma

Conclusion: Six1 Can Regulate Metastasis in RMS Cells

Highly metastatic Poorly metastatic

Six1

-Actin

Validation ofDifferentialExpression

(RNA)

Six1 expression enhances metastasisin poorly metastatic RMS cells

Blocking Six1 function inhibits metastasis in highly metastatic RMS cells

The Role of Six1 in Metastasis: Validation and Functional Studies

P < 0.001

Ezrin (ERM family: Ezrin-Radixin-Moesin)

Multiple passmembrane proteins Single pass

membrane proteins

Activation by PIP2 bindingand phosphorylationby Rho and PKC

Signaling Functions•Activates Rho

•Activates PI3K/Akt•Substrate of c-MET

Nature review

Ezrin

-Actin

Highly metastatic Poorly metastatic

P < 0.01

Validation ofDifferentialExpression

(Protein)

Ezrin expression enhances metastasisin poorly metastatic RMS cells

Blocking Ezrin function inhibits metastasis in highly metastatic RMS cells

The Role of Ezrin in Metastasis: Validation and Functional Studies

Conclusion: Ezrin Can Also Regulate Metastasis in RMS Cells

Expression of both SIX1 and EZRIN is significantly elevatedin advanced human RMS

Kruskal Walis One-Way NonParametric ANOVA

Conclusions:

•SIX1 and EZRIN may play a role in human RMS progression

•The regulation of SIX1 and EZRIN expression may be connected

• Microarray analysis identified a set of genes whose expression was significantly different between highly and poorly metastatic RMS cells.

• Forced expression of Ezrin or Six1 significantly enhanced metastasis in poorly metastatic cells, while disruption of Ezrin or Six1 function inhibited metastasis in highly metastatic cells.

• EZRIN and SIX1 expression levels were elevated in human RMS tissue, significantly correlating with progression.

• The identification of EZRIN and SIX1 as critical regulators of metastasis in RMS provides new mechanistic and therapeutic insight into this childhood cancer.

Summary/Conclusions

• We identified Ezrin and Six1 as critical regulators of metastasis in rhabdomyosarcoma (RMS).• Both Ezrin and Six1 can regulate metastasis. • Metastatic consequence of Ezrin and Six1 expression are Similar.• Ezrin and Six1 appear to be coordinately expressed. Yu, et al. Nature Medicine, 2004; 10: 175-181

• What is the relationship between Ezrin and Six1?

Six1 regulates transcription of the ezrin gene

Q: How does Six1 regulate the expression of Ezrin?H: Six1 directly regulates the transcription of Ezrin gene

Six1

Six1 CC siRNA

RMS772 RMS14

Ezrin-actin

Low level Ezrinlow level Six1Poorly metastatic

high level Ezrinhigh level Six1highly metastatic

Six1 can bind to Ezrin promoter

Chromatin Immunoprecipitation (ChIP)•IP with anti-Six1 antibody•PCR with ezrin gene promoter primers

An

ti -

Six

1

IgG

No

an

tib

od

y

Neg

ativ

e

Mar

ker

-1106 to -870

-230 to -121

-1106 to

-870

0

0.5

1

1.5

2

2.5

3

3.5

0.20.20.20.20.2

siRNA 0.20.1 0.1 0.2 0.3 0.40.3 0.4

Six10.50.40.30.20.1(g)

-1616 LUC

0

1

2

3

4

5

Rel

ativ

e lu

cife

rase

act

ivit

y

Six1 can activate the Ezrin promoter and stimulate expression of reporter

gene

Six1 can bind to MEF3-like motif in Ezrin promoter

Six1 (g) 0 0.5 1 2

Wild type

0 0.5 1 2

Deleted

0

1

2

3

4

5

Rel

ativ

e lu

cife

rase

act

ivit

y

TCAGG

LUC-1106 -870

TCAGGIgG

Six1

Hot probe

Cold probe

Antibody

+

+ ++ +

+

++

+ + +

ccccaatagaaattcaggagcagctcg-1087 -1061

• Six1 can bind to Ezrin gene promoter and regulate Six1 can bind to Ezrin gene promoter and regulate the transcription of Ezrinthe transcription of Ezrin

• Ezrin is a downstream gene of Six1Ezrin is a downstream gene of Six1

H: Inhibiting Ezrin expression blocks some function of Six1

Blocking Ezrin by siRNA can abrogate the Six1 induced metastasis in vivo

No.

gro

ss lu

ng

met

asta

sis

siEzrin

Six1_+

++

++_

_

0

5

10

15

20

25

30

Six1

Ezrin

-actin

Tail vein injection

RMS772

Conclusion: Six1 promotes metastasis through the transcriptional activation of Ezrin

• Six1 can bind to Ezrin gene promoter and regulate Six1 can bind to Ezrin gene promoter and regulate the transcription of Ezrinthe transcription of Ezrin

• Ezrin is a downstream gene of Six1Ezrin is a downstream gene of Six1

• Blocking Ezrin by siRNA can abrogate the Six1-Blocking Ezrin by siRNA can abrogate the Six1-induced metastasis in vivo induced metastasis in vivo

Q: What is the mechanism?

RMS772 RMS119 RMS14

3H-t

hym

idin

e in

corp

orat

ion

0

10000

20000

30000

40000

50000

60000

70000

80000

C Six1 C Six1 C Six1

P < 0.005

P < 0.001

P < 0.01

Six1 stimulates the proliferation of RMS cells

C Six1 C Six1 Six1 Six1

RMS14 RMS772

Six1 can stimulate the phosphorylation of Erk1/2 and AKT.

P – Erk1/2

Erk1/2

P - AKT

AKT

- actin

Six1

Disrupting Ezrin by siRNA blocks effect of Six1 on phosphorylation of AKT, but not Erk1/2.

- actin

P - AKT

AKT

P – Erk1/2

Erk1/2

- +

Six1

siEzrin+-+++-

Ezrin

Six1

-actin

Both Six1 and Ezrin can enhance ECM attachment in RMS cells

_

Six1Ezrin

_ +__+

Per

cen

t att

ach

men

t(30

min

)

0

10

20

30

40

50

60

70

80

*

* p < 0.05

Per

cent

att

ach

men

t(30

min

)

_++

+ ++siEzSix1 _

_

0

10

20

30

40

50

60

70

80

90

* p< 0.05

**

Blocking Ezrin by siRNA can inhibit ECM attachment ability of Six1 in RMS cells

Ezrin

Six1

AKTErk

CellProliferation

CellSurvival

Cell Motilityand Adherence

Rho-family

siRNA

summarysummary

• Six1 can bind to Ezrin gene promoter and regulate the Six1 can bind to Ezrin gene promoter and regulate the transcription of Ezrintranscription of Ezrin

• Blocking Ezrin by siRNA can abrogate the Six1 induced Blocking Ezrin by siRNA can abrogate the Six1 induced metastasis in vivometastasis in vivo

• Both Six1 and Ezrin can enhance ECM adhesion in RMS Both Six1 and Ezrin can enhance ECM adhesion in RMS cellcell

• Blocking Ezrin by siRNA can inhibit Six1-mediated ECM Blocking Ezrin by siRNA can inhibit Six1-mediated ECM adhesion in RMS cellsadhesion in RMS cells

• Knocking down Ezrin reversed the stimulatory effects Knocking down Ezrin reversed the stimulatory effects of Six1 on Ezrin expression and Akt activity, but had of Six1 on Ezrin expression and Akt activity, but had little effect on expression of Six1, or on MAPK activitylittle effect on expression of Six1, or on MAPK activity

Yu, et al. Cancer Research, 2006

The transcriptional control• Regulation of transcription factor Six1

• Modification of chromatin/histone

Histone modification patterns can define gene activity

K9 K27

K4 K9S10

K14R17

K18 K23 K36

K79

Histone H3

Histone H3

ACTIVE

INACTIVE (silenced) Acetylation (lysine)

Methylation (lysine, arginine)

Phosphorylation (serine)

Hypothesis: Modification of chromatin/histone is associated with

transcriptional regulation of Ezrin

- 11 kb3 kb4 kb5 kb6 kb7 kb8 kb 2 kb

1 2 3 4 5 6

RMS14high level Ezrin

RMS772 low level Ezrin

0

0.20.4

0.60.8

1

1.21.4

1.6

1 2 3 4 5 6

K4M

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6

K9A

00.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6

K9M

RMS14

RMS772

Histone patterns in Ezrin chromatin are consistent with an active configuration

Chromatin Immunoprecipitation (ChIP) Analysis of Histone Modifications at the Ezrin Locus

K9M K9M

K4M K4M

K9A K9A

<>>

ChIP assay•IP with histone H3 modification-specific antibodies Di-methyl-lysine 9 (K9M) Tri-methyl-lysine 4 (K4M) Acetyl-lysine 9 (K9A)•PCR with Ezrin gene primers

Conclusion : Histone modifications at the ezrin locus are linked to Ezrin expression

The HDAC inhibitor reactivates Ezrin expression

0 24

48

TSA 300 nM

18s rRNAEzrin

RMS772

RMS14

hours12 72 96M

An

ti -

Six

1

IgG

No

an

tib

od

y

Neg

ativ

e

Mar

ker

Six1 can bind to Ezrin chromatin/histone modification locus

Chromatin Immunoprecipitation (ChIP)•IP with anti-Six1 antibody•PCR with ezrin gene primers (locus 6)

Suggestion: Six1 may associate with chromatin/histone remodeling proteins

in regulation of Ezrin gene

Six1 binding region is same as the highly active locus in Ezrin chromatin

Six1

CB

P

IP

WB

Six1

CBP

IgG

Six1 can interact with CBP

Suggestion: Six1 can recruit CBP to Ezrin promoter locus

Hypothesis

Six1 can associate with CBP therefore regulating Ezrin expression.

CBP, a chromatin/histone remodeling protein, has acetyltransferase (HAT) activity, which acetylates histone and other proteins

Rel

ated

act

ivit

y of

luci

fera

se

(x 1

0000

)

0

5

10

15

20

25

30

35

pEP-Luc

+Six1 _ _+ ++_ _

CBP-GFP _ _ +_ _ __ +

_+CBP WT _ _ _

+_

_ _CBP∆HAT __++

_ __

Suggestion: CBP negatively participates in Six1-mediated regulation of Ezrin expression

* p < 0.002

Rel

ated

act

ivit

y of

luci

fera

se

(wit

h S

ix1/

wit

hou

t Si

x1)

DMSO

200 nM TSA

300 nM TSA

500 nM TSA

TSA TSA TSA TSA

pEP-Luc

Six1 0 g 0.05 g 0.1 g 0.2 g

1

2

3

4

5

6

7

8

9

10

0

Acetyltransferase (HAT) negatively regulates the Sxi1-mediated regulation of Ezrin gene

CBPAc

EzrinSix1

??

Summary

• Six1 can bind to Ezrin promoter and regulate Ezrin expression

• Histone modification patterns in Ezrin chromatin are consistent with an active configuration and linked to Ezrin expression

• Six1 can associate with CBP therefore regulating Ezrin expression

• CBP has a dual effect on the regulation of Ezrin gene

Pathway Assessment of Metastasis and Preclinical Modeling

Cutaneous Malignant Melanoma

Hepatocyte Growth Factor/Scatter Factor (HGF/SF)

and Malignant Melanoma

•HGF/SF Binds to the Receptor Tyrosine Kinase c-MET Proto-oncoprotein

•Aberrant HGF/SF-MET Signaling Is Implicated in Cutaneous Malignant Melanoma

•Chromosome gains are frequently observed at the c-MET locus in melanoma

•HGF/SF-MET autocrine loops are evident in most human melanoma cell lines

•c-MET is overexpressed in many human melanoma samples

•c-MET overexpression negatively correlates with overall patient survival

•HGF/SF-MET Signaling Regulates Many Processes Associated with Oncogenesis

(proliferation, migration, invasiveness, survival, angiogenesis)

HostCancer cell

The successful establishment of metastasis

Interaction

B16, 37-7 cells

c-Met

HGF/SF

NK2

c-Met + HGF/SF

c-Met + NK2

HGF/SF + NK2

Transgenic mouse models

HGF/SF

NK2

Nude mice

FVB

BL6

Paracrine/endocrine Met signaling can enhance metastasis in vivo

Autocrine & paracrine/endocrine signaling are equally effective in stimulating metastasis

With HGF/SF, NK2 super-induced the metastasis of melanoma cells

Summary• Overexpression in tumor cells of Met receptor alone did not

stimulate Met-related activities either in vitro or in vivo.

• However, as expected, the creation of an autocrine loop through co-expression of HGF/SF and Met facilitated growth, motility and invasiveness in vitro, and experimental metastasis in vivo.

• Notably, metastasis of Met-expressing tumor cells placed in a transgenic host overexpressing HGF/SF (creating paracrine/endocrine stimulation) was at least as high as autonomous receptor stimulation in the same cells.

• HGF/SF and its splice variant NK2 were not functionally interchangeable, either in vitro or in vivo.

• Alone, NK2 stimulated tumor cell motility and invasiveness but not growth in vitro, and failed to facilitate experimental metastasis.

• But, in conjunction with HGF/SF, NK2 super-induced the metastasis of tumor cells.

Yu Y and Merlino G. Cancer Res. 2002

PTENPI3K

PI4,5P2

PI3,4,5P3

pAKT

Bcl2

Caspase

GSK-3FKHR

pAKT

pRB

p27

Cyclin E

CDK2

FAK ShcGrb2

Sos

pMEK

pMAPK

To die apoptosis

To sleep Cell cycle arrest

To regulate cell adhesion, migration

Goal: To understand how PTEN regulates tumor

cell spreading and motility as well as metastasis

Phosphatase activity

Prot

ein

Lip

id

Cel

l cy

cle

Akt

inhi

biti

on

Mig

rati

on in

hibi

tion

WT 1 403Cat C2 PDZ/BD

L -- - ±GE mutant

P - - - - -CA mutant

Rel

ativ

e M

otil

ity

1

2

3

4

5

6

0

7

C

LY

WT

WT

P L

Rel

ativ

e In

vasi

vene

ss

0

1

2

3

4

C

LY

WT

WT

P L

Endogenous PTENGFP-PTEN

C WT

WT

P L

No.

Gro

ss L

ung

Met

asta

sis

C

WT

PLP

shR

NA

C1

WT

PLP shR

NA

C2

Endogenous PTENGFP-PTEN

WT

Pre Labeled In Vitro 1hr Lung 24 hr Lung6 hr Lung

PTEN WT

PTEN P

PTEN L

B16

Hypothesis: PTEN protein phosphatase acts downstream on substrate(s) to suppress metastatic behavior in melanoma cells

Cell extract

phosphoprotein

2D electrophoresis

Differential spots

Mas-spec

Novel substrates of the PTEN protein phosphatase in metastasis pathways by protemics

The combination of two images from 2D gel electrophoresis of phosphoproteins purified from wild-type PTEN (green) and phosphatase-catalytically-deficient PTEN (magenta) melanoma cells.

Acknowledgments

Glenn MerlinoRichard Sharp

National Cancer InstituteJaved KhanChand KhannaLee Helman

National Human Genome Research InstitutePaul Meltzer