Download 2012CTOS_Takahashi

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 Medicine
4 Gastroenterology Center, The Cancer Institute Hospital of Japanese Foundation of
Cancer Research
Dept. Tissue Regeneration
Kyoto Univ.
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?
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 as a metastasis-related gene
Background:
in spindle cell sarcomas
MFH
LMS
SS
PLS
MPNST
UDS
65 STS resected specimens
metastasis (+) n=29
(-) n=36
Human mesenchymal stem cell (reference)
cDNA microarray
Genes involved in
distant metastasis
Isolation of AFAP1L1
(Furu et al. Oncogene 2011)
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 as a metastasis-related gene
Background:
in spindle cell sarcomas
Relative expression of AFAP1L1
STS cases with metastases
100
**
** *
** * *
10
0
STS cases without metastases
100
**
MFH
LMS
SS
LS
MPNST
UDS
* Relative value > 100
(Furu et al. Oncogene 2011)
10
0
High incidence of distant metastasis in AFAP1L1-positive
Dept. Tissue Regeneration
tumors irrespective of type of tumors.
Kyoto Univ.
Background:
AFAP1L1 expression is significantly
associated with distant metastases
Variables
Comparison
P-value
Surgery
Chemotherapy
Diagnosis
Female vs male
≧60 vs <60
Extremities vs trunk
Primary vs recurrence
≧5cm vs 5cm>
Superficial vs deep
Grade 3 vs others
Wide vs others
Performed vs not performed
MFH vs others
0.7173
0.0251
0.4840
0.6578
0.2826
0.6640
0.0103
0.9004
0.2482
0.3795
AFAP1L1
Positive vs negative
0.0021
Univariate analyses
Gender
Age
Location
History
Size
Depth
FNCLCC
Multivariate analyses
Age
FNCLCC
AFAP1L1
Cox’s proportional
hazards model
<Endpoint >
The occurrence of
distant metastases
(Furu et al. Oncogene 2011)
0.0365
0.0075
0.0021
Dept. Tissue Regeneration
Kyoto Univ.
Relative expression of AFAP1L1
1000
P = 0.0093
100
10
Metastasis-free fraction (%)
Background: AFAP1L1 as a prognostic marker for STSs
AFAP1L1 (-)
P = 0.0058
AFAP1L1 (+)
Follow-up period (years)
(-)
(±)
(+)
(++)
1
Mets (-)
(n=23)
Mets (+)
(n=18)
(Furu et al. Oncogene 2011)
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 expression positively
Background:
correlates with cell invasiveness in vitro
Gelatin zymogram
(Furu et al. Oncogene 2011)
AFAP1L1 may promote matrix invasion via MMP-9 secretion.
Dept. Tissue Regeneration
Kyoto Univ.
Research question
What is the molecular mechanism of AFAP1L1
in the invasive or metastatic phenotype?
Dept. Tissue Regeneration
Kyoto Univ.
Methods
Immunocytochemistry
- Change of morphology by AFAP1L1
- Subcellular localization of AFAP1L1
Xenografting
- Effect of AFAP1L1 expression in vivo
Immunoprecipitation
- Associating protein
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 induces actin-rich dots,
and localizes in the structure
F-actin
AFAP1L1
AFAP1L1
AFAP1L1
Mock
F-actin / DAPI
10um
10um
Merge
Dept. Tissue Regeneration
Kyoto Univ.
Structure of invadopodia
SaOS2
/AFAP1L1
Actin rich core
Merge
Vinculin
F-actin
10um
Adhesive ring
(Upper: Yamaguchi et al. Biochem Biophys Acta 2007)
(Lower: Yilmaz et al. Cancer Metastasis Rev 2009)
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 induces gelatin-degrading invadopodia
Fluorescence-conjugated
gelatin
Gelatin-AF488
Merge
AFAP1L1
Mock
F-actin
20um
X-Z
Defect
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 localizes in the ring structure of invadopodia.
SaOS2 / AFAP1L1
AFAP1L1
Vinculin
Merge
10µm
5um
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 accelerates tumor growth of sarcoma cells
(Furu et al. Oncogene 2011)
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 accelerates tumor growth of carcinoma cells
RKO
Parental
pLenti6 /
AFAP1L1
AFAP1L1
β-actin
10000
AFAP1L1
3
Tumor Volume (mm3)
Cell Numbers (×106)
β-actin
Parental
LacZ
2
AFAP1L1
1
1
2
3
4
LacZ
8000
AFAP1L1
6000
*
4000
2000
0
0
0
* P = 0.008
Parental
5
Day
0
1
2
3
4
5
6
Week
Dept. Tissue Regeneration
Kyoto Univ.
siRNA local administration suppresses tumor growth
Control siRNA
AFAP1L1 siRNA
Control AFAP1L1
siRNA
siRNA
AFAP1L1
β-actin
RKO/AFAP1L1
A2 clone
(Approx. 100mm3)
3,000
Untreated
No
treat
Control siRNA
AFAP1L1 siRNA-3
2,000
P = 0.009
1,000
0
10
15
20
25
30
siRNA administration
35
Day
Tumor Weight (mg)
Tumor Volume (mm3)
P = 0.002
3,000
2,000
1,000
0
Control
AFAP1L1
siRNA
siRNA-3
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 regulates cellular morphology and motility
RKO
***
***
80%
80
40%
40
0.8
*
0.6
0.4
0.2
A2
AFAP1L1
A2
AFAP1L1 siRNA-4
LacZ
AFAP1L1
Untreated
Control
siRNA
AFAP1L1
siRNA-3
% Rounded Cells
-80
**
80
0.8
40
0.4
AFAP1L1 siRNA-1
control siRNA
50µm
00
No treat
Untreated
A2
(µm)
AFAP1L1 siRNA-3
80
-80
**
0
LacZ
0%0
Parental
80
(µm / min)
Parental
AFAP1L1
(Clone A2)
50µm
Mean Speed
LacZ
AFAP1L1
(Bulk)
% Rounded Cells
Parental
*P<0.05; **P<0.01; ***P<0.001
AFAP1L1
β-actin
Dept. Tissue Regeneration
Kyoto Univ.
Rounded cells form fewer focal adhesions in RKO cells
F-actin / Vinculin / DAPI
Elongated
Round
RKO
Flattened
10µm
Block MR. et al. Eur J Cell Biol. (2008)
Dept. Tissue Regeneration
Kyoto Univ.
Vinculin regulates cell shape and motility
• F9 (mouse embryonic carcinoma cells)
Round phenotype
Vinculin -/-
Smaller focal adhesion complex
Cell motility ↑ (on 2D substrate)
Rodriguez Fernandez et al. J Cell Biol. (1992)
• MEF (mouse embryonic fibroblast)
Vinculin
-/-
Cell-matrix adhesion ↓
Cell motility ↑ (on 2D substrate)
Xu et al. J Cell Sci. (1998)
Dept. Tissue Regeneration
Kyoto Univ.
Hypothesis
AFAP1L1
Negative modulation ?
Association ?
Mature focal adhesion complex
Vinculin
Cell-matrix adhesion
Flattened or elongated phenotype
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1 associates with vinculin
150 100 75 -
(Flag)3AFAP1L1
IB: Flag
50 150 100 -
IP: AFAP1L1
IgG
kDa
IP: Vinculin
LoVo
IP: HA
IgG
kDa
WCL
RKO /
(Flag)3-AFAP1L1
+
(HA)3-Vinculin
150 100 75 -
Vinculin
IB: Vinculin
50 -
IB: HA
100 -
IB: AFAP1L1
Dept. Tissue Regeneration
Kyoto Univ.
Vinculin regulates anoikis
Parental
LacZ
AFAP1L1
3
2
Parental
LacZ
AFAP1L1
10000
8000
6000
4000
1
2000
0
0
1
2
3
4
5
0
0
Vinculin↓
→
1
2
3
4
5
6
anoikis↓
(Subauste et al. J Cell Biol 2004)
Anoikis: apoptosis induced by lack of
correct cell-matrix attachment
Dept. Tissue Regeneration
Kyoto Univ.
AFAP1L1-transduced cells are resistant to anoikis
(kDa)
150 -
LacZ
(2-Hydroxyethyl methacrylate)
AFAP1L1
HEMA coating
Parental
RKO
Full length PARP
100 -
Cleaved PARP
75 -
Suspension culture
AFAP1L1
ACTB
possibly the cause of accelerated tumor growth in vivo
Dept. Tissue Regeneration
Kyoto Univ.
Conclusion
What is the molecular mechanism of AFAP1L1
in the invasive or metastatic phenotype?
# 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.
Dept. Tissue Regeneration
Kyoto Univ.
Acknowledgement
Toguchida-lab
Makoto Ikeya
YongHui Jin
Akira Nasu
Kazuo Hayakawa
Sakura Tamaki
Elalaf Hassan
Kyosuke Kobayashi
Yoshihisa Matsumoto
Koji Yokoyama
Makoto Fukuda
Sho Hineno
Naoko Takahara
Chie Koizumi
Michiko Ueda
Yukiko Kobayashi
Hisayo Yasuda
Marie Yoshino
Institute for Medical Science,
University of Tokyo
Toyomasa Katagiri
Yusuke Nakamura
Seiya Imoto
Hidetoshi Shimohira
Department of Orthopaedic Surgery,
Kyoto University
Koichi Nishijo
Tomoki Aoyama
Dept. Tissue Regeneration
Kyoto Univ.
Conclusion
What is the molecular mechanism of AFAP1L1
in the invasive or metastatic phenotype?
# 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.
Dept. Tissue Regeneration
Kyoto Univ.