Dia 1

Role of the IGF-1/IGF-1
receptor pathway in the 5TMM
experimental mouse model

IGF-1 is a survival, proliferative and migratory factor
in multiple myeloma
Georgii-Hemming P et al Blood 1996
Jelenik DF et al 1997
Vanderkerken et al Blood 1999
Ge NL et al Blood 2000
Ferlinet et al Br J Haematol 2000
Qiang YW et al Blood 2002
Mitsiades CS et al Oncogene 2002
Standal T et al Blood 2002
Podar K et al J Biol Chem 2003
Tai YT et al Cancer Res 2003
Qiang YW et al Blood 2004
Abroun S et al Blood 2004
Menu E et al Br J Cancer 2004
Mitsiades CS et al Cancer Cell 2004
Descamps G et al J Immunol 2004

5TMM model
Spontaneous development in old
C57BlKaLwRij mice
The model is maintained by in vivo transfer in
young syngeneic C57BlKaLwRij mice
Preferential localization in hematopoietic
organs (bone marrow and spleen)
Ig serum concentration is correlated to tumor
mass
Myeloma induced angiogenesis in the BM
Radl J et al, J Immunol. 122, 609-613, 1979
Vanderkerken K et al, Br J Cancer 76, 451-460, 1997
Van Valckenborgh et al. Br J Cancer 86 (5), 796-802, 2002

IGF-1/IGF-1 R in 5TMM cells
Effect of IGF-1 on migration, proliferation and VEGF
secretion
Altered gene expression after stimulation with IGF-1
Regulation of IGF-1 receptor expression
by the bone marrow microenvironment
Effect of a specific IGF-1 RTK inhibitor on tumor
development in the 5T33MM model

IGF-1 induces migration of 5TMM cells, mediated
by a PI3K pathway
60
50
)
% 40
(
n
30
r
a
t
i
o
g 20
mi
10
0
0
5
10
IGF-1 concentration in ng/ml
K. Vanderkerken et al. Blood 93: 235-241 (1999)
E.Menu et al Ann. N.Y. Acad.Sci. 973: 124-135 (2002)
Menu E et al. Br J Cancer 90: 1076-1083 (2004)

IGF-1 induces DNA synthesis mediated by a
PI3K/Akt-MEK/ERK pathway
Menu E et al. Br J Cancer 90, 1076-1083 (2004)

IGF-1 induces VEGF secretion mediated
by a MEK/ERK pathway
Menu E et al. Br J Cancer 90, 1076-1083 (2004)

Activation of the IGF-1 R on 5T33MM cells

PI3 kinase
Raf

IRS
Akt
MEK
Erk 1/2
F-actin polymerization
DNA synthesis
VEGF secretion
Menu E et al. Br J Cancer 90, 1076-1083, 2004

Altered gene expression after stimulation
of 5T33MMvv cells with rIGF-1: A Microarray approach
430A2.0 array
Upregulated: 29 genes
Metabolic function
Protein synthesis
Signaling
Microtubuli
Regulation of expression
Downregulated: 27 genes
Apoptosis
Protein degradation

Expression of IGF-I receptor on 5T33MMvv and
5T33MMvt and migration towards IGF-1
5T33MMvv
5T33MMvt
IGF-IR IGF-IR
Asosingh et al. Cancer Research 60,3096-3104, 2000

Expression of IGF-I receptor -chain on
5T33MM vitro cells 18h after injection
Asosingh et al. Cancer Research 60,3096-3104, 2000

Migration of 5T33MMvtvv towards IGF-I
60
40
20
% migration 0
0 5 10
IGF-I (ng/ml)

Upregulation of IGF-I receptor after contact
with BM endothelial cells
IGF-I receptor

IGF-1/IGF-1 R in 5TMM cells
Effect of IGF-1 on migration, proliferation and VEGF
secretion
Regulation of IGF-1 receptor expression
by the bone marrow microenvironment
Altered gene expression after stimulation with IGF-1
Effect of a specific IGF-1 RTK inhibitor on tumor
development in the 5T33MM model

Aim
To investigate the role of inhibiting the IGF-1 receptor tyrosine
kinase by Picropodophillin (PPP), in the 5T33MM murine model
PPP,member of the cyclolignan family, interferes with the IGF-1
receptor autophosphorylation at the substrate level.
in vitro experiments
in vivo experiments
Girnita A et al, Cancer Research 64, 3:236-242, 2004

PPP reduces the phosphorylation of the IGF-1R
and downstream signaling
A
120
n
100
y
l
a
t
i
o
l
)
80
phor
t
r
o
n
60
f
co
t
ophos
o
u
(%
40
a
1
R
F-
20
I
G
0
0
0,5
1
1,5
2
2,5
PPP (然)
B

PPP reduces IGF-1 induced F-actin polymerization
F-actin
1,6
*
**
1,4
1,2
1
I
control
0,8
MF
PPP
0,6
0,4
0,2
0
control
IGF-1
p < 0,04
E.Menu et al Ann. N.Y. Acad.Sci. 973: 124-135 (2002)

PPP reduces IGF-1 induced DNA synthesis
DNA synthesis
n 2,5
*
*
a
t
i
o
or 2
r
p
c
o
n
i 1,5
ne
control
i
di
PPP
1
y
m
h
0,5
r
e
l
a
t
i
ve t
0
control
IGF-1
p < 0,02

PPP reduces IGF-1 induced VEGF secretion
control
QuickTimeTM and a
Photo - JPEG decompressor
are needed to see this picture.
VEGF secretion
2,5
*
*
e
x 2
d
n
i
n
i
o 1,5
5T33MM
et
control
1
PPP
0,5
r
e
l
a
t
i
ve secr
0
QuickTimeTM and a
Photo - JPEG decompressor
control
IGF-1
are needed to see this picture.
p < 0,05

Effect of treatment with PPP
on tumor burden and angiogenesis
in the 5T33MM Model
0 weeks
4 weeks
5T33MM model
Inject 5T33MM cells
Sacrifice
Myeloma Disease
Paraprotein
FACS
Continuous PPP (7 or 20 mg/kg, twice a day, or vehicle) in 5T33MM Angiogenesis
MVD

Assessment of tumor load
paraprotein
Idiotype staining
4
*
**
100
*
**
3,5
90
M
80
3

B
70
2,5
l)
60
/d
2
a cells in
50
(
g
m
1,5
40
elo
y
30
1

m
20
0,5
%
10
0
0
naive
vehicle
low PPP
high PPP
naive
vehicle
low PPP
high PPP
p< 0,0001
p< 0,0001

Assessment of microvessel density
Vehicle
microvessel density
35
***
****
30
25
D 20
MV 15
10
PPP
5
0
naive
vehicle
low PPP
high PPP
P < 0,02

Effect of treatment with PPP
on survival
in the 5T33MM Model
0 weeks
4 weeks
5T33MM model
Survival:
Inject 5T33MM cells
Kaplan-Meier analysis
Continuous PPP (20 mg/kg, twice a day, or vehicle) in 5T33MM

Effect of high dose PPP on survival of the mice
P < 0,001

Conclusion

PPP

DNA synthesis
VEGF secretion
tumor
tumor load
angiogenesis
survival
Menu E et al, submitted

General conclusion
IGF-1 R is expressed by the 5TMM cells and the
expression level is regulated by the bone marrow
microenvironment
IGF-1/IGF-1R binding results in the activation of two
distinct signaling pathways
IGF-1/IGF-1R binding results in proliferation,
migration of the MM cells and in increased VEGF
secretion
Blocking IGF-1/IGF-1R binding results in decreased
proliferation and VEGF secretion and is associated
with decreased tumor burden and increased survival
of the mice

Free University Brussels
Helena Jernberg-Wiklund, PhD
Prof. Dr. Ben Van Camp, MD, PhD
Thomas Stromberg, MD, PhD
(Uppsala University, Sweden)
Ivan Van Riet PhD
Marleen Bakkus PhD
Rik De Raeve, MD, PhD
Kewal Asosingh, PhD
(UIA, Antwerp, Belgium)
Els Van Valckenborgh
Eline Menu
Peter Croucher, PhD
Elke De Bruyne
(Sheffield University, UK)
Jo Caers, MD
Tomas Bos
Evy De Leenheer
Isabelle Vanden Broek, MD
Angelo Willems
Carine Seynaeve
Ronald De Zanger

QuickTimeTM and a
GIF decompressor
are needed to see this picture.