Genes and proteins of myeloma endothelial cells to
searchi
h spec f
hi i
ific
ifi targe
t
ts
t
f
o th
the
th tumor
t
vasculature
t
Angelo Va
V cca, MD, PhD
Department of Internal Medicine
and Clinical Oncology
University of Bari Medical School
Bari, Italy
XII International Myeloma
My
Wo
W rkshop
o
Wh
Was
W
i
Wh
hi
t
ng on DC
D
DC
26 February--11st March 2009
Neovessel sprouting in patients with active multiple myeloma
(FVIII
(FVIII--RA
RA staining)
Myeloma
MGUS
Benign anemia
Va
V cca et al. Br J Haematol, 1994
Ultrastructural findings of MM endothelial cells
C
G
A
C
E
F
H
B
D
Va
V cca et al. Blood 2003
In vitro and in vivo angiogenic activity of bone marrow endothelial
cells of patients with multiple myeloma
Matrigel 8 hr
MM endothelial cells
HUVECs
x25
CAM 4 days
C
d
on iti
diti
d
one
di
me um of MM
MM endoth li
e l
a
l
ce llls
bFGF
Pt. 1, diagnosis
Pt. 2, relapse
x5
**
**
*
MM endothelial cells (MMECs) express markers indicative
of both an angiogenic and a vasculogenic state
Mesenchymal stem cells
Hemangioprecursors
gp
HUVECs
MMECs
VEGF-
VEGF R2
(CD34+CD133+)
PDGF-R
PDGF-R
Plasma
+ PDGF-BB
Indicative of:
cells
CD133
VEGF-
VEGF R2
PDGF-R
R
a
li
vascul
vascu ogeni
ogen c
i
PDGF-R
VEGF
state
Angioblasts
VEGF-R2
Endothelial cells
VEGF-
VEGF R1
Stromal cells
VEGF-R2
an angiogenic
Tie2/TEK
state
Tie2
Ang-1
FVIII-RA
PDGF-R
PDGF-BB
Pericytes
Va
V cca et al. Blood 2003;
Va
V cca et al., Blood 2006
Mature Vascular Network
Coluccia - Vacca,
Vacca, Blood 2008
Rolny et al. Blood 2006
Ria Vacca,
Vacca, Clin Cancer Res, 2008
cDNA microarray for MM endothelial cells. A report on 10 patients ( )
and 3 newborns' HUVECs ( )
cytokines,
y, chemokines and
40
growth factors and receptors
40
growth factors and receptors
80
35
adhesion molecules
30
70
25
60
APDH
APDH
G 20
G
%
*
*
%
*
10
*
10
*
* * *
*
*
*
*
*
*
0
0
k
1
R1
R2
R3
1
bFGF
FGF-7
VEGF-A
VEGF-B
VEGF-C
VEGF-D
GRO
TGF-
TGF-
TGF-
TGF-
EDG1
Tie2/Te
Neuropil-1
IFN-
PRL
SPARC
TNF-
TNFSF15
ITGAV
CD31
matrix, proteases and inhibitors
other angiogenesis-related genes
150
125
*
20
100
*
75
15
*
DH
DH
20
P
PA
*
A 10
*
G
G
10
%
%
*
*
5
*
*
*
0
0
(PLAU)
Fibronectin-1
MMP-2
MSR-1
uPA
PEDF
Thrombosp-1
Thrombosp-2
TIMP-1
TIMP-2
ETS-1
HIF-1
ID3
CD105
RSN
Osteopont
DNA microarray analysis of various endothelial cell types
VEGF
bFGF
y
800
y
800
250
600
200
intensit
intensit
150
400
100
200
50
Flurescence
0
Flurescence
0
Active
Activ
Kaposi
HUVECs
Active
Activ
Kaposi
HUVECs
MMECs
MMECs
HGF
IGF-1
150
sity
100
sity
80
ten
ten
in
60
in
100
ce
ce
en
40
en
50
esc
20
esc
ur
ur
Fl
0
Fl
0
Active
Kaposi
HUVECs
Active
Kaposi
HUVECs
MMECs
MMECs
IGFBP-3
Ang1
ity 5000
ity
250
4000
200
intens 3000
intens
150
2000
100
1000
50
Flurescence
0
Flurescence
0
Active
Kaposi
p
HUVECs
Active
Kaposi
HUVECs
MMECs
MMECs
Active
MMECs
MGECs
HUVECs
KS
# 1 2
3
45# 1 2
3
45# 1 2
3
4
5 #1
bFGF
VEGF
Ang1
A
HGF
Va
V cca et al, JCO 2005;23:5334-46
IGF-1
IGFBP-3
Expression levels of VEGF165 and SEMAPHORIN-3A in MGUS
and MM endothelial cells in comparison with paired plasma cells
Real Time RT-PCR
VEGF165
SEMAPHORIN-3A
MGUS
MM
MGUS
MM
15.0
15.0
P < .02
EC
P < .03
EC
12.5
12.5
of
of
P < .05
10.0
10.0
7.5
7.5
ression
ression
p
p
P < .05
p
5.0
5.0
ex
ex
2.5
2.5
Fold
Fold
0
0
MGECs
Plasma
MMECs
Plasma
MGECs
Plasma
MMECs
Plasma
cells
cells
cells
cells
Western Blot
VEGF165
SEMAPHORIN-3A
MGUS
MM
MGUS
MM
kDa
kDa
22
117
15
MGECs Plasma MMECs Plasma
MGECs Plasma MMECs Plasma
cells
cells
cells
cells
Va
V cca et al, Blood 2006; 108: 1661
1661--77
The link between Hypoxia
Hypoxia--Inducible
Inducible Factor (HIF)
(HIF)--11
and VEGF is Ras
Other angiogenesis-related
genes in MM endothelial cells
20
*
15
10
Low pH
GAPDH% 5
%
*
Hypoxia
0
RAS
ETS-1
HIF-1
ID3
CD105
RSN
GTP
Osteopont
Ras
Ras--V12
V12
Raf
MEK
ERK 1/2
1/2
Von
V
Hippel HIF-1
HIF-1
GCG
Lindau
CGCGC
Xu et al, Oncogene 2005
Expression of HIF
HIF--11 by MM endothelial cell (MMECs), but
not MGUS endothelial cells (MGECs) and HUVECs
MGECs (Pt. 12)
MMECs (Pt. 9, relapse)
MMECs (Pt. 14, refractory)
Constitutive autophosphorylation
kDa
205
VEGFR-2p
50
ERK-2p
HUVECs (sample #21)
37
Ria - Vacca,
Vacca, unpublished results
HIF
HIF--11 + alexa488 + Ulex
Ria et al., Thromb Haemost 2004
Plasma cells and endothelial cells
in MM patients share sy
pymmetric TK receptors
PDGF
VEGF
PDGF
VEGF
VEGF
VEGF--R2
R2
VEGF
VEGF--R2
R2
PDGF
PDGF--R
R
PDGF
PDGF--R
R
Plasma
Endothelial
cell
cell
Src
Akt
Src
Akt
VEGF
Erk 1/2
PDGF
VEGF
Erk 1/2
PDGF
Proliferation
Chemotaxis
Survival
Coluccia Vacca,
Vacca, Blood 2008
Expression of vessel
vessel--associated
associated cellular markers by MM
endothelial cells (MMECs) and HUVECs
(FACS
(F
and We
W stern
e
blot
blot analyses)
80
P < 0.0001
70
60
P < 0.001
VECsU 50
UH 40
P < 0.0001
15
of 10
P < 0.001
P < 0.01
sion
5
s
P < 0.01
4
3
P < 0.05
P < 0.05
P < 0.05
expresd 2
d
Fol
1
0
lin
34
1
n
R
s
Tek
R-1
R-2
R-3
R-1
R-2
R-3
g
D
D31
133
n
ctin
g
rin
c
erin
F
g
an-1
di
o
F
C
C
C
C
a
C
n
pE
ie2/T
VEG
inte
VEGF
VEGF
bFGF
sele
bFGF
bFGF
ndec
Endo
E
3
HUVE
Aquapori
VE-cadh
sy
CD133,A
CD105,
D62E,
D61,C
144,
138,
hrombospo
C
C
D
D
T
DC
C
Samples tested:
tested MMECs = 37 to 57; HUVECs = 11
1 to 22.
Significance
Significan
by Fisher and Kruskal
Kruskal--Wallis
Wallis test followed by
Va
V cca et al, Blood 2003;102:3340
2003;102:3340--88
Duncan (t), Bonferroni (t), and Wilcoxon
W
paired tests.
Studies on isolated tumor-derived endothelial cells
Origin
Sorting Ag
Findings
Culture
Publication
hu Colon carcinoma
CD146
9T
9 ECs
TECs
-
St Croix et
et al
al Science
Science
2000
hu Colon carcinoma
CD31, CD34
17 TECs
-
Van Beijnum et al Blood
2006
hu Ovarian
CD146
23 TECs
-
Lu et al Cancer Res
carcinoma
CD31/CD146 Laser
12 TECs
2007; Buckanovich et al
J Cli
Clin O
l
nco 2007
hu Mammary/Colon
CD105
13 TECs
-
Seaman et al Cancer
carcinomas
Cell 2007
hu Renal Breast
Breast
CD105
Expression of
+
Bussolati et al FASEB J
carcinomas
embryonic
embry
genes &
2003
oncogenes;
Grange et al Oncol Rep
Rep
genetic instability
2006
hu Melanoma
CD31 BS1-B4
Chromosomal
+
Hida et al Cancer Res
Liposarcoma
Liposarco
lectin
translocations &
2004; Amin et al Cancer
Liposarcoma
Liposarco
multiple mutations
Res 2006
hu B
B--cell
cell
CD31
Chromosomal
Chromosomal
+
Streubel et al N Engl J
Ly
L mphoma
y
abnormalities
Med 2004
cDNA microarray analysis (Affymetrix) of
188 genes up-regulated
MM endothelial cells (MMECs) vs. HUVECs
in MMECs vs HUVECs
Unsupervised analysis
Supervised analysis
(856 genes)
(366 genes)
HUVECs
MMECs
HUVECs
MMECs
178 genes down-regulated
# 1
2
34
5
1
23
4
5
# 1
2
3
4
5
1 2
3
4
5
in MMECs vs HUVECs
R. Ria et al., CCR, in revision
Genes up-regulated in MM endothelial cells vs. HUVECs
8152 metabolism (52)
[0.7% of 7080]
7582
hi
physi l
o
i
og
l
ca
processes (88)
[0.8% of 10745]
8151 cell growth and/or
maintenance (34)
[0.8% of
of 4181]
Biological
9987
7154 cell communication
7165 signal
process
cellular process (69)
(41) [1.0% of 3788]
transduction (25)
[0.9% of 7385]
[0.8% of 3044]
7275 development (37)
9653 morphogenesis (25)
9887 organogenesis (24)
[1.9% of 1942]
[]
[2.2% of 1116]
[]
[2.2% of 1059]
Unsupervised analysis
Searching genes specifically
MMECs vs. MGECs
Supervised analysis
distinguishing
MMECs vs. MGECs
MMECs vs. MGECs
22 genes
down
12000
10000
8000
6000
COL4A1
4000
2000
0
1
2
3
_4
_9
_2
_3
_4
_5
EC_1
EC_2
EC_3
EC_4
EC_8
MM_
MM_
MM_
MM
MM
S_1
US
US
US
US
HUV
HUV
HUV
HUV
HUV
MGU
MG
MG
MG
MG
12000
10000
up
8000
COL6A3
6000
COL6A1
4000
2000
0
_3
_8
1
2
3
4
9
_1
_3
_4
EC_2
EC
EC_4
EC
MM_
MM_
MM_
MM_
MM_
US
US_2
US
US
US_5
UVEC_1
UV
UV
UV
UV
MG
MG
MG
MG
MG
HUV
HUV
HUV
HUV
HUV
MG
MG
MG
MG
R. Ria et al., CCR, in revision
Va
V lidation
a
of MMEC and MGEC genes and proteins by
real
real--time
time RT
R --PCR
PCR and We
W stern
e
blot
d
Hypoxia regulated
down
d
up
Hypoxia-
Tu
T mor
u
suppressor
protein.
DIRAS3
gene. Down--regulated
regulated
BNIP3
Overexpressed in
n
n
1
4
in ovarian and breast
breast cancer, and
0.75
NSCLC
3
cancer
ressio
ressio
p
0.50
p
2
xp
ex
e
0.25
1
0
Fold
0
Fold
MMECs
MGECs
MMECs
MGECs
MMECs
MGECs
MMECs
MGECs
Selenoprotein W
SERPINF1
(antioxidant) in
Anti
Anti--angiogenic
angiogenic
SEPW1
n
response to
10000
activity by interacting
n
1
oxidative stress
8000
with VEGFR
VEGFR--11
ressio
0.75
(protective)
6000
ressio
xp
4000
0.50
xp
e
2000
e
0.25
Fold
0
0
Fold
MMECs
MGECs
MMECs
MGECs
MMECs
MGECs
MMECs
MGECs
Tu
T mor
u
suppressor gene.
Regulator of cell
It induces apoptosis in
growth and
SRPX
human cancer cell lines.
IER3
apoptosis. High
n
1
Down--regulated
regulated in human
n
3
levels in many
0.75
prostate and colon cancer
2.5
tumor cell lines
ressio
essio
0.50
2
exp
0.25
1.5
expr
0.5
0
Fold
0
Fold
MMECs
MGECs
MMECs
MGECs
MMECs
MGECs
MMECs
MGECs
One dimensional SDS
SDS--page
page of cell lysates of HUVECs, MGECs,
and MMECs (*refractory,
refractory °relapse
relapse))
MGECs
MMECs°
PROTEIN
HUVECs
kDa
MMECs*
280
-FILAMIN
200
180
140
120
103
-ACTININ1
105
HSP90
100
BUB 1
90
GLUCOSE REGULATED
PROTEIN PRECURSOR
70
NANOELECTROSPRAY
NANOELECTROSPRA
ANNEXIN VI
(MS/MS)
55
VIMENTIN
50
-ENOLASE
40
25
ANNEXIN V
20
PROTEIN ZETA/ DELTA
Berardi Vacca,
Vacca, unpublished results
Confirmatory We
W stern
e
blot of selected proteins in MM endothelial
cells (MMECs), MGUS endothelial cells (MGECs) and HUVECs
MM
MM
MM
Diagnosis Progression MM
Relapse
HUVECs MGECs
MGECs
Refractory
kDa
280
-FILAMIN
120
HIF-1
1
Heat Shock Protein 90
83
(HSP 90)
70
ANNEXIN VI
8
5
VIMENTIN
28
PROTEIN ZETA/DELTA
-ACTIN
40
DIFFERENTIALLY EXPRESSED PROTEINS IN MMECs vs. MGECs AND HUVECs
FUNCTIONAL
PROTEIN
CLUSTERING
EXPRESSION
EXPRESSIO IN
IN MMECs
MW
-ENOLASE
METABOLISM
UPREGULATED
47KDa
-ACTININ 1
STRUCTURAL
UPREGULATED
103KDa
PROTEIN
ANNEXIN V
COAGULATION
UPREGULATED
50KDa
ANNEXIN VI
SIGNAL
UPREGULATED
70KDa
TRANSDUCTION
(active phase)
-FILAMIN
STRUCTURAL
UPREGULATED
280KDa
PROTEIN
GLUCOSE REGULATED
REGULA
CHAPERONE
UPREGULA
U
TED
LA
78KDa
PROTEIN PRECURSOR
PROTEIN
HSP 90
CHAPERONE
UPREGULATED
83KDa
PROTEIN
BUB1
CELL GROWTH
DOWNREGULATED
120KDa
MAINTENANCE
PROTEIN ZETA/DELTA
CELL GROWTH,
UPREGULATED
28KDa
MAINTENANCE
VIMENTIN
STRUCTURAL
UPREGULATED
58KDa
PROTEIN
Genes expressed by MM vs. MGUS endothelial cells
MMP activation
Spreading
Collagen
Morphogenesis Collagen
Sprouting
Anchorage
CRY
CR A
Y B
A
Anchorage
SERPINF1, POSTN
Growth
COL4A1
COL6A1, COL6A1/A3,
C
COL6A1/A3,
PRG1
TNC, PCOLCE, ASPN,
Collagen
EGFR
CTSK
Anchorage
Growth
LOX, LUM
Growth
VEGF
HIF
HIF--11
LDB2 Omeostasis
SEPW1
Chemotaxis
KRT7
BNIP3
Spreading
Anti
Anti--apoptosis
apoptosis
CCL2 (MCP
(MCP-1)
CXCL12 (SDF
(SDF--11)
Up
Up--regulated
regulated
IER3
Down
Down--regulated
regulated
Anti
Anti--apoptosis
apoptosis
Gene
NOTCH
SRPX
Growth
DIRAS3, GEM
Function
HSPB7
TGF
TGF--
Apoptosis
Anti
Anti--proliferation
proliferation
Stress
Signaling
R. Ria et al., CCR, in revision
Several features of MM endothelial cells
Common Precursor
seem to point to a tumoral endothelial cell
Ferrata's
Ferrata' (tumor) cell
inside the MM bone marrow
Tu
T mor
u
Tum
Tu o
m r
o hemangioblast
h
hemangioblast ?
Unknown yet
Is myeloma a plasma cell tumor
plus an angiosarcoma?
MM
MM
endothelial
cell
Plasma cell
T o
w sisters?
MM
Plasma cell
Fusion
(immortalization of MGUS endothelial cells)
Parent 1
Parent 2
Daughters
Hybrids?
Many thanks!
R. Ria (Hemangioprecursors),
M. Boccadoro, M. Massaia, Hematology, Torino
F. Bussolino, Biochemistry, Torino
A. Basile (PTX-bFGF)
P. Corradini, Hematology, Milan
S. Berardi (proteomics)
N. Di
Di Renzo
Renzo, Hematology, Lecce
C. De Luisi (genomics)
P. Di Tonno, Hematology, Bari
G. Di Pietro (macrophages),
A. Guarini, Hematology IRCCS, Bari
Dept. Internal Medicine and Clinical
P. Musto, Hematology, Rionero, Potenza
Ol
Oncology, Bari, IT
IT
A. Neri, Hematology, Milan
M.T. Petrucci, M.R. Ricciardi, Hematology, Rome
Head: Prof. F. Dammacco
M. Presta, Biotechnology, Brescia
K. Anderson, Dana Farber, Boston
D. Ribatti, Anatomy, Bari
H. Dvorak, Dept. Anatomy, Boston
B. Nico, Anatomy, Bari
D. McDonald, Dept. Anatomy, Los Angeles
A. Roccaro, Dana Farber, Boston
M. Coluccia, A. Boccarelli, Pathology, Bari
Special thanks to Associazione Italiana per la
la Ricerca
Ricerca sul Cancro
Cancro (AIRC
(AIRC, Milan)