Induction of Multi-Lineage Markers and Their Down-
regulation by IL-6 in Human Myeloma cells
Michio M. Kawano, MD,PhD
Lb
Lab
t
ora ory of Cl
Ce lll l
u ar Si
Si
l
gna A
l
na ysis
Graduate School of Medicine
Yamaguchi University
gy
Ube, JAPAN
XI IMW, Kos Island, Greece, 2007.6.26
Background
· Myeloma cells from myeloma patients show
"phenotypic heterogeneity"; immature, intermediate
dt
and mature myeloma celllls.
( Kawano MM, et al. Blood 82:564,1993)
· Most myeloma cells
cells express CD56,
CD56 and some myeloma
cells express CD33 (phenotypic plasticity).
(Lanier LL, et al. J Exp Med 69:2233,1989)
· Ml
Myeloma
l
cells lose th
the expression f
o
t
mas er gene f
o B
cell linage, PAX-5 (lineage instability).
(Mahmoud MS, et al. Blood 87:4311,1996;
Nutt SL, et al. Nature 401:556,1999)
· Myeloma cells have various types of chromosomal
translocation with 14q (genetic
(
instability).
instability
(Avet-Loiseau H, et al. Blood 99:2185,2002)
Phenotypic
Heterogeneity &
Plasticity
·Morphological heterogeneity
·myelod/monocytoid
(immature, mature myeloma cells)
CD33+
·Very
Very few mitosis
mitosis
Phenotypic plasticity (Lineage Instability)
CD138
CD56
CD11b
CD13
IgG
CD34
CD33
CD86
CD61
BFU-E
Erythroid
Myeloid
CFU-Meg
HSC
HSC
Meg
CFU-GM
T/NK
T
Lymphoid
NK
Pre-B
GC-B
BM-plasma cells
PAX-5
Immature
mature
B cell lineage
PAX-5
RAG, AID
IL-6
Various types of chromosomal translocation
(genetic Instability)
t(11;14)
del(13)
CCND1
p53 inactive
t(4;14)
FGFR3
MMSET
t(14;20)
t(14;16)
t(6;14)
MAFB
t(14;16)
t(6;14)
c-MAF
MUM1
Question
Question
Why
do myeloma cells show such
phenotypic heterogeneity and plasticity?
Myeloma cells express multi-lineage markers without PAX-5 expression.
MM1
MM2
MM3
MM4
MM5
A.
33
7
56
4
86
D
D
D
D
D
D
D
C
C
C
C
C
CD38
B
ILKM8
ILKM3
NOP2
AMO1
MSG-Y01
MSG-Y01
B
ILKM8
0
99
01
CD4
CD33
C
CD4
CD86
CD7
CD4
CD86
CD56
C
ILKM8
NOP2
AMO1
Y01
KUS
Raji
MM1 MM2 MM3 MM4 MM5
Pax 5
G3PDH
D
33
33
7
56
CD
CD
CD
CD
CD7
CD56
CD56
CD86
Liu 01
Some myeloma cells express CD33 with increased C/EBP.
A
B U266
Liu01
E
IgG
x1,000
x1,000
Vector
Vector
Vector
000
Vector
CD33
C/EBP
C/EBP
C/EBP
U266
IgG
U266
KUS
U266
ILKM3
CD33
Liu01
C
U266
Liu01
1
1/10
1
1/10
cDNA
IgG
F
ILKM3
Liu01
CD33
CD33
hi
ve
l
c e
hi
ve
l
c e
ILKM3
Vit D3
Vit. D3
C/EBP
IgG
PU.1
Count
CD33
ILKM8
ETS-1
CD33
G3PDH
Raji
IgG
vehicle
Vit. D3
0 0
0 0
CD33
98 2
99 1
KUS
D
1
U266
Liu0
ILKM8
ILKM3
KUS
Raji
CD33
IgG
C/EBP
CD33
Raji
CD7
G3PDH
CD56(+) myeloma cells express neuronal cell markers.
A
-Y01
nt
1
1
2
u
B
MM1
MM2
MM3
MM4
MM5
MM6
MM7
u
G
6
G
0
O
M3
M8
S11
K
K
P
K
co
Y01
ILKM3
MS
U26
Liu
AM
IL
IL
NO
KM
Nestin
Cell
NSE
Nestin
NSE
-tubulin III
U266
ILKM8
-tubulin III
Chromogranin A
Chomoganin A
G3PDH
Liu01
NOP2
G3PDH
AMO1
KMS11
CD56
MM 6 (x400)
D
U266(x400)
NOP2(x400)
1
C
266
iu0
MO1
KM8
OP2
US
U
L
A
L
A
I
N
K
NSE
46 kDa
44 kDa
-actine
Control IgG
Anti-NSE
Anti-NSE
NSE-producing myeloma
myeloma cells
· Nakajima T,
T, Noguchi
Noguchi T, Kumahara Y,
Y, et
et al.
Neuron specific enolase-producing IgD
multiple my
pyeloma with high
g serum
amylase activity. Rinsho Ketsueki
1995;36:359-364.
· Jimbo J, Sato K, Ikuta K, et al. A neuron
specific enolase-producing multiple
l
mye oma. Rinsho K t
e
k
sue i
ki 2006 47
;
1381
:
-
1386.
CD56(+) myeloma cells express CD7 with NK cell activity.
A
Vehicle
B
Forskolin
+ IL-6
U266
Count
U266
Vehicle
Forskolin
+ IL-6
AMO1
Liu01
Vehicle
Forskolin
+ IL-6
ILKM8
CD7
U266(Forskolin +IL-6)
U266
C
U266
Liu01
(Forskolin+IL-6)
E
11/10
1 1/10
1 1/10
cDNA
Perforlin
MEF
G3PDH
U266
KUS
Raji
D
3
1
0 0
0
0
33
60 40
98 2
D
99 1
D
99
C
CD7
Human myeloma
myeloma cells
1) CD56(+)
() ~ 90%
· CD56(+) with neuronal cell markers(NSE,
nestin, -tubulin III, chromog
,granin A)
~ neuronal cell
· CD56(+) with CD7(+) ~ NK cell
2) CD33(+) ~ 10%
CD33(+)CD13(-) ~ myeloid/monocytoid
progenitor
IL-6 down-regulates the expression of CD33 and CD56.
U266
Liu01
D
B
Control
Control
-+
-
+
IL-6
A
IL-6
IL-6
(10 ng/ml for 5 days)
Liu01
C/EBP
50
Liu01
)
PU.1
40
U266
(%
CD56
CD7
30
G3PDH
cells
sitive
20
o
Liu01
Liu01
Liu01 wi
with
th IL-
IL 6
U266
U266
p
C
10
CD33
0
03
6
9
12 Days
x400
x400
x400
E
70
Medium
F
U266
Liu01
60
*
IL-6
0 10 30
0 10 30
IL-6 (min)
50
%
STAT3-p
40
ecells
30
STAT3
sitiv
20
Po
Erk1/2--p
*
10
0
Erk1/2
CD33
CD56
Induction of multi-lineage markers and
their down-regulation by
gy IL-6
( Liu S, et al. Int J Hematol 85:49, 2007)
(Hypothesis)
GC B cells
Immature Plasma Cell
Mature Plasma Cell
CD19
B cell lineage
CD45
Loss of
PAX-5
Dropped out of the B cell lineage
CD19(-)
PAX-5(-)
IL-6
Proliferation (Self-renewal)
"Stone cells" stem cell-like cells dropping o to
ut ft
of he
the line
li
ag
Differentiation (multi-lineage)
Myeloma
myeloid, NK cell, neuronal cell
( Liu S, et al. Int J Hematol 85:49, 2007)
Self-renewal
Lineage commitment
Lineage maintenance
Stem cell
Cancer stem cells ?
Differentiated cells
Progenitor
Loss of expression of master gene(s)
dropping out of the lineage
Multi-lineage
differentiation
Stem cell-like cancer cells ?
Stone cells
(Stem cell like cells
dropping out of the lineage)
Myeloma cells are "Stem cell-like Cancer cells" ?
We
We call them
them " Stone cells";
stem cell-like cells dropping out of the lineage.
Contribution
· Liu S, Abroun S, Otsuyama K, Shamsasenjan K,
Amin J, Islam A, Iqbal MS
(Lab. Cellular Signal
(g
Analysis
y
, Graduate School of Medicine,
Yamaguchi Univ.)
· Asaoku H, Iwato K
(4th Dept.
(p Internal Medicine, Hiroshima Red Cross Hospital
p
)
· Sakai A
(Dept. Hematology and Oncology, Hiroshima Univ)
· Otsuki T
(Kawasaki Medical School)
· Shimizu S
(Shimane Pref. Hospital)
· Tsuyama N
( Hiroshima Univ)