p62-
p
A Potential Target
g
for
Blocking Microenvironmental
Support of Myeloma
G. David Roodman MD PhD
VA Medical Center and
University of Pittsburgh
Hideshima T et al Nat Rev Cancer. 2007,8:585-98.
10 Stromal Cells Derived from MM
Patients Increase Myeloma Cell
Growth and IL-6 Production
Normal Stromal
Cells
(pg/ml)
)4
MM Patient Stromal
0
Cl
Ce llls
200
8
*
(x1
*
*
150
ulture
6
C
/Culture
/C
100
4
Number
roductionp 50
Cell
2
IL-6
0
0
0
*, p<0.05 compared
MM1.S
ANBL6
Normal MM
with normal stromal
cells
Adhesive Interactions between Myeloma Cells
and Marrow
Marrow Stromal
Stromal Cells Increase
NFkB and p38 MAPK Signaling
Myeloma Cell
NfKB
NfKB
p38
Stromal Cell
The NFkB and p38 MAPK Signaling are
Activated in Normal and MM
MM Stromal
Stromal Cells
Treated with TNF-
Normal
MM Patients
0
1
2
5
10
15
0
1
2
02
5
10
10
10
15
15
15
(Min.)
p-
p IB
B
IB
-actin
Normal
MM Patients
(Mi
(
)
0
5
10
15
20
30
0
5
10
15
20
30
(Min.
0
5
10
15
20
30
0
5
10
15
20
30
(Mi
p-p38 MAPK
p38 MAPK
MAPK
-actin
Increased VCAM-1 Expression is Blocked by
the NEMO Binding Protein,
g, but not a
p38MAPK Inhibitor
Normal
MM Patient
TNF-
TNF-
TNF-
TNF-
TNF-
TNF-
+
+
+
+
+
+
NEMO
DMSO
NEMO
NEMO
DMSO
NEMO
Control
TNF- -inhibitor
(Neg)
Control
TNF- -inhibitor
(Neg)
VCAM-1
-actin
VCAM-1/
ti
-ac n
1.0
5.2
2.6
5.1
4.8
1.0
1.
6.6
2.7
5.9
6.0
Normal
MM Patient
TNF-
TNF-
+
+
Control
TNF- SB203580 SB203580
Control
TNF-
SB203580 SB203580
VCAM-1
-actin
VCAM-1/-actin
1.0
4.5
4.0
1.1
1.0
6.0
5.3
1.1
IL-6 Production in Stromal Cells Induced by
TNF- is Blocked by a p
yp38 MAPK Inhibitor
but not NEMO Binding Protein
IL-6 (pg/ml)
Normal Stromal
1200
Cells
MM Patient
1000
Stromal Cells
800
* p<0.01 compared
with TNF-
TNF alone.
600
*
400
200
*
0
TNF- (10
/
ng
l)
m
­
+
+
­
+
SB203580 (10M) ­
­
+
+
­
NEMO-biding peptide (10mM) ­
­
­
­
+
p62 Is an Adapter Protein Involved in
Multiple Signaling Pathways
Pathways
P62
Survival
Growth and Differentiation
Growth/Differentiation
Structure of
of p62
p62
PB1
ZZ
p38
TBS
UBA
PB1: aPKC binding domain
ZZ: RIP binding domain
p38: p38 MAPK binding domain
TBS: TRAF6 binding domain
UBA: Ubiquitin binding domain
p-PKC / PKC Expression is Increased
in Stromal Cells from MM Patients
MM Patients
Normals
p62
phospho-PKC
1
2
3
4
1
2
1.4
*
p62
4
1.2
p62/-actin
1.2
1.1
1.0
1.0
1.1
1.0

1.0
KC
pPKC
n
3

P/
0.8

-acti
PKC
/ 0.6
2
p62
ho-PKC
pPKC
pPKC /PKC
/PKC
40
4.0
30
3.0
60
6.0
24
2.4
15
1.5
10
1.0
p
0.4
1
actin
phos
0.2
0
0
Normals MM
Normals MM
Knockdown of p62 in Stromal Cells
Decreases Both NFkB and p38MAPK
p38MAPK
Signaling in Response to TNF-
Control siRNA
p62 siRNA
pNFkB
-
NFkB
GAPDH
Control siRNA
p62 siRNA
0
5
10 15 20 30
0
5
10 15 20 30 (Mi
(
)
n.
p-
p-p38MK
p38MAPK
p62 Knockdown in Myeloma Stromal Cells Decrease
p-
p PKCz and VCAM-
VCAM 1 Protein
Protein Levels
Normal
MM Patient
Control
p62
Control
62
p
siRNA siRNA
siRNA siRNA
p62
62-kDa
p62/-actin
1.0
0.1
1.0
0.1
p-PKC
80-k
- Da
pPKC-/-actin
10
1.0
01
0.1
10
1.0
02
0.2
PKC
80 -k
- Da
PKC-
C /-
actin
1.0
0.
00 3
1.0
0.2
0
VCAM-1
110
11 -k
- Da
VCAM-1/-actin
1.0
0.3
1.0
0.3
-actin
43-k
- Da
Knockdown of p62 in Stromal Cells
Decreases IL-6 Production
Production and Support
Support
of MM Cell Growth
MM Cell Growth in Co-Cultures
IL-6l
6 l
l
eve s in Co-Cl
Cu t
ltures
(x104)
* ; p<0.01 compared
(pg/ml)
with control siRNA
* ; p<0.01 compared
transduced cells
re
with control siRNA
8
600
e
u
transduced cells
u
500
6
/Cult
400
ion
er/Cultur
*
4
300
*
*
200
ellNumb 2
6product
*
C
IL- 100
0
0
CTL-
CTL
p62
CTL-
CTL
p62-
p62
CTL-
CTL
p62
CTL-
CTL
p62-
p62
siRNA -siRNA
siRNA
siRNA
siRNA -siRNA
siRNA siRNA
Normal
MM Patient
Normal
MM Patient
Stromal Cells Stromal Cells
Stromal Cells
Stromal Cells
p62 is Important for Stromal Cell Support
of Human Multiple Myeloma Cell Growth
* p<0.01
300
re
200
s/Cultu
Cell 100
M1SM
0
p62+
p
/-
p62
p
-/-
Without
Stromal calls
OCL Formation is Decreased in Co-Cultures
of Normal OCL Precursors
Precursors with
p62-/- Stromal Cells Treated with TNF-
150
p62+/- Stromal Cells
p62 -/- Stromal Cells
e
* , p<0.05 compared with WT
stromal cells
100
NC/CulturM
ositive
50
P
*
TRAP
0 Vehicle
TNF-
(100 pg/ml)
RANKL Production by p
yp62+/- and
p62-/- Stromal Cells
3000
p62+/- Stromal Cells
p62 -/- Stromal Cells
2500
)
* , p<0.01 compared with
2000
p62+/- stromal cells
(pg/mlL 1500
1000
RANK
500
*
*
0
Ct
TNF
Cont
TNF-
(100 pg/ml)
p62-/- Mice Have Normal
Nb
Numbers f
o CFU
CFU GM
-
Conclusions
· p62 in stromal cells plays an important role in
the formation of multiple complexes that
activate distinct sil
igna iling pathways
hi
w ch
increase stromal cell support of tumor growth
d
an ostl
teoclast ft
forma i
tion in MM.
· These results suggest that p62 is an attractive
target for treating multiple myeloma.
Acknowledgements
N Kurihara
D Galson
Y Hiruma
J Anderson
Funding: VA Merit Review and MMRF