Outline

Dept. Tissue RegenerationKyoto Univ.

AFAP1L1 accelerates growth of sarcoma cells

in vivo through the association with

protein complex in invadopodia

Ryo Takahashi1,2, Yoichiro Kajita1, Tomohisa Kato1, Moritoshi Furu1, Masaya Ikegawa3, Satoshi Nagayama4, Yoshiharu Sakai2

and Junya Toguchida1

1 Dept. Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto Univ.2 Dept. Surgery, Graduate School of Medicine, Kyoto Univ.3 Dept. Genomic Medical Sciences, Graduate School of Medical Sciences, Kyoto Prefectural Univ. of Medicine4 Gastroenterology Center, The Cancer Institute Hospital of Japanese Foundation of Cancer Research


Outline

Background:AFAP1L1 : Actin filament-associated protein 1-like 1

- isolated as a metastasis-related gene

in soft tissue spindle cell sarcomas

(STSs)

- involved in the invasion of sarcoma cells in vitro.

Research question:- What is the molecular mechanism of AFAP1L1

in the invasive or metastatic phenotype?


Background:

65 STS resected specimensmetastasis (+) n=29

(-) n=36

Genes involved in distant metastasis

Isolation of AFAP1L1

AFAP1L1 as a metastasis-related genein spindle cell sarcomas

MPNST

MFH LMS SS

PLS UDS

Human mesenchymal stem cell (reference)

cDNA microarray

(Furu et al. Oncogene 2011)


Background:

MFHLMSSSLSMPNSTUDS0

10

100 * * * * * * * * *

0

10

100 * *

STS cases with metastases

STS cases without metastases

Rel

ativ

e ex

pres

sion

of A

FAP

1L1

High incidence of distant metastasis in AFAP1L1-positive tumors irrespective of type of tumors.

AFAP1L1 as a metastasis-related genein spindle cell sarcomas

* Relative value > 100



　 Variables Comparison P-value

Univariate analysesGender Female vs male 0.7173 Age ≧60 vs <60 0.0251 Location Extremities vs trunk 0.4840 History Primary vs recurrence 0.6578 Size ≧5cm vs 5cm> 0.2826 Depth Superficial vs deep 0.6640 FNCLCC Grade 3 vs others 0.0103 Surgery Wide vs others 0.9004 Chemotherapy Performed vs not performed 0.2482 Diagnosis MFH vs others 0.3795 AFAP1L1 Positive vs negative 0.0021

Multivariate analysesAge 0.0365 FNCLCC 0.0075

　 AFAP1L1 　 0.0021

Background: AFAP1L1 expression is significantlyassociated with distant metastases

<Endpoint >The occurrence of distant metastases

Cox’s proportional hazards model



(-) (±) (+) (++)

Follow-up period (years)M

etas

tasi

s-fre

e fra

ctio

n (%

)

AFAP1L1 (+)

AFAP1L1 (-)

P = 0.0058

AFAP1L1 as a prognostic marker for STSsBackground:

(n=23) (n=18)

1

10

100

1000

Mets (-) Mets (+)

Rel

ativ

e ex

pres

sion

of A

FAP

1L1 P = 0.0093



Background:

Gelatin zymogram


AFAP1L1 expression positivelycorrelates with cell invasiveness in vitro

AFAP1L1 may promote matrix invasion via MMP-9 secretion.


Research question

What is the molecular mechanism of AFAP1L1



Methods

Immunocytochemistry - Change of morphology by AFAP1L1

- Subcellular localization of AFAP1L1

Xenografting - Effect of AFAP1L1 expression in vivo

Immunoprecipitation - Associating protein


Moc k

AFAP

1L1

F-actin / DAPI

10um

AFAP

1L1

F-actin AFAP1L1

Merge10um

AFAP1L1 induces actin-rich dots,and localizes in the structure


Structure of invadopodia

(Upper: Yamaguchi et al. Biochem Biophys Acta 2007)(Lower: Yilmaz et al. Cancer Metastasis Rev 2009)

F-ac

tinV

incu

lin

SaOS2/AFAP1L1

Mer

ge

10um

Actin rich core

Adhesive ring


AFAP1L1 induces gelatin-degrading invadopodia

Moc

kAF

AP1L

1

F-actin Gelatin-AF488 Merge

20um

X-Z

Fluorescence-conjugatedgelatin

Defect


AFAP1L1 localizes in the ring structure of invadopodia.

AFAP1L1 Vinculin Merge10µm

SaOS

2 / A

FAP1

L1

5um


AFAP1L1 accelerates tumor growth of sarcoma cells



AFAP1L1 accelerates tumor growth of carcinoma cells

0 1 2 3 4 50

1

2

3 ParentalLacZAFAP1L1

Cell

Num

bers

(×10

6 )

Day

AFAP1L1

Pare

ntal

pLe n

t i6 /

LacZ

Clo n

e A

2

Bul k

pLenti6 /AFAP1L1

RKO

β-actin

AFAP1L1β-actin

0 1 2 3 4 5 60

2000

4000

6000

8000

10000ParentalLacZAFAP1L1

Tum

or V

olum

e (m

m3 )

Week

* P = 0.008

*


siRNA local administration suppresses tumor growth

AFAP1L1β-actin

ControlsiRNA

AFAP1L1siRNA

P = 0.002

Tum

or W

eigh

t (m

g)Control siRNA

AFAP1L1 siRNA-3

0

1,000

2,000

3,000

AFAP1L1 siRNA

RKO/AFAP1L1A2 clone

(Approx. 100mm3)

Control siRNA

P = 0.009

siRNA administration

Day

Tum

or V

olum

e (m

m3 )

10 15 20 25 30 35 0

1,000

2,000

3,000 No treatControl siRNAAFAP1L1 siRNA-3

Untreated


AFAP1L1 regulates cellular morphology and motility

(µm)

80-80

-80

80

50µmParental

AFAP1L1(Bulk)LacZ

RKOAFAP1L1(Clone A2)

Par

enta

l

LacZ

AFA

P1L

1

A20%

40%

80%

% R

ound

ed C

ells

80

40

0

******

Mea

n S

peed

Par

enta

l

LacZ

AFA

P1L

1

A20

0.2

0.4

0.6

0.8 ***(µm / min)

*P<0.05; **P<0.01; ***P<0.001

UntreatedControlsiRNA

AFAP1L1siRNA-3

A2

50µm

AFAP1L1

β-actin

No

treat

cont

rol s

iRN

A

AFA

P1L

1 si

RN

A-1

AFA

P1L

1 si

RN

A-3

AFA

P1L

1 si

RN

A-4

0

0.4

0.8

% R

ound

ed C

ells

80

40

0

**

Unt

reat

ed


Rounded cells form fewer focal adhesions in RKO cells

RoundFlattened Elongated

F-actin / Vinculin / DAPI

10µm

RK

O

Block MR. et al. Eur J Cell Biol. (2008)


Vinculin regulates cell shape and motility

• F9 (mouse embryonic carcinoma cells)

Vinculin -/-

Round phenotype

Smaller focal adhesion complex

Cell motility ↑ (on 2D substrate)

• MEF (mouse embryonic fibroblast)

Cell-matrix adhesion ↓

Cell motility ↑ (on 2D substrate)

Rodriguez Fernandez et al. J Cell Biol. (1992)

Xu et al. J Cell Sci. (1998)

Vinculin -/-


Hypothesis

VinculinMature focal adhesion complex

Cell-matrix adhesion

Flattened or elongated phenotype

AFAP1L1

Negative modulation ?Association ?


AFAP1L1 associates with vinculin

(Flag)3-AFAP1L1

IB: Flag

IB: HA

RKO / (Flag)3-AFAP1L1

+(HA)3-Vinculin

100 -150 -

IgG

IP: H

A

WCL

75 -

50 -

100 -

150 -kDa

IB: Vinculin

Vinculin

IB: AFAP1L1

LoVo

IgG

IP: A

FAP1

L1

IP: V

incu

lin100 -

75 -

50 -

100 -

150 -kDa


Vinculin regulates anoikis

0 1 2 3 4 5

0

1

2

3 ParentalLacZAFAP1L1

0 1 2 3 4 5 6

0

20 00

40 00

60 00

80 00

10 00 0 ParentalLacZAFAP1L1

Anoikis: apoptosis induced by lack of correct cell-matrix attachment

Vinculin↓ → anoikis↓ (Subauste et al. J Cell Biol 2004)


AFAP1L1-transduced cells are resistant to anoikis

HEMA coating (2-Hydroxyethyl methacrylate)

Suspension culture

possibly the cause of accelerated tumor growth in vivo

ACTB

Full length PARP

Par

enta

l

AFA

P1L

1

75 -

100 -

150 -(kDa) La

cZ

AFAP1L1

Cleaved PARP

RKO


Conclusion



# AFAP1L1 modulates cell-matrix interaction;

1. assembly of invadopodia,

2. cell shape and motility,

# AFAP1L1 plays a role in the inhibition of anoikis,

presumably through the interaction with vinculin.


Acknowledgement

Toguchida-lab

Makoto IkeyaYongHui JinAkira NasuKazuo HayakawaSakura TamakiElalaf HassanKyosuke KobayashiYoshihisa MatsumotoKoji YokoyamaMakoto FukudaSho HinenoNaoko TakaharaChie KoizumiMichiko UedaYukiko KobayashiHisayo YasudaMarie Yoshino

Institute for Medical Science,University of Tokyo

Toyomasa KatagiriYusuke NakamuraSeiya ImotoHidetoshi Shimohira

Department of Orthopaedic Surgery,Kyoto University

Koichi NishijoTomoki Aoyama


Conclusion



# AFAP1L1 modulates cell-matrix interaction;

1. assembly of invadopodia,

2. cell shape and motility,

# AFAP1L1 plays a role in the inhibition of anoikis,

presumably through the interaction with vinculin.

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