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Crucial role of phosphatase CD45 in determining signaling and proliferation of human myeloma cells

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In multiple myeloma, a large number of growth factors (IL-6, IGF-1, FGF, HGF and HB-EGF) are involved in promoting myeloma cell growth. In the present study, a serum-free, cytokine-free, collagen-based assay, which does not allow the generation of spontaneous myeloma colonies, was used to identify the clonogenic growth factors for fourteen myeloma cell lines. IL-6 is the only clonogenic factor able to stimulate both CD45+ and CD45- myeloma cell lines, generating myeloma colonies from 10 out of 14 myeloma cell lines. Using a pharmacological Erk inhibitor, we show that the Erk/MAPK pathway is involved in IL-6-induced clonogenicity of CD45+, but not CD45- myeloma cell lines. In contrast to IL-6, the other growth factors (IGF-1, FGF, HGF and HB-EGF) stimulate only some myeloma cell lines, but always CD45-, and less effectively than IL-6. Among them, IGF-1 is the most potent, generating myeloma colonies from five out of eight CD45- myeloma cell lines. Finally, the capacity of IGF-1 and FGF to stimulate the clonogenicity of CD45- myeloma cells correlates with their ability to stimulate the Erk/MAPK pathway. We conclude that CD45 expression plays a crucial role in determining signaling and proliferation of human myeloma cell responses to IL-6, IGF-1 and other growth factors. The poor outcome of CD45- myeloma patients could be related to the capacity of CD45-myeloma cells to take advantage of multiple growth factors.
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UNCORRECTED PROOF
Crucial role of phosphatase CD45 in determining signaling
and proliferation of human myeloma cells
Madeleine Collette
1
, Géraldine Descamps
1
, Catherine Pellat-Deceunynck, Régis Bataille,
Martine Amiot
INSERM, U601, Département de Recherche en Cancérologie, LNC Label, Institut de Biologie, 9 quai Moncousu, 44000 Nantes, France
Correspondence : M. Amiot
<mamiot@nantes.inserm.fr>
ABSTRACT. In multiple myeloma, a large number of growth factors (IL-6, IGF-1, FGF, HGF and HB-EGF) are
involved in promoting myeloma cell growth. In the present study, a serum-free, cytokine-free, collagen-based
assay, which does not allow the generation of spontaneous myeloma colonies, was used to identify the clonogenic
growth factors for fourteen myeloma cell lines. IL-6 is the only clonogenic factor able to stimulate both CD45+
and CD45- myeloma cell lines, generating myeloma colonies from 10 out of 14 myeloma cell lines. Using a
pharmacological Erk inhibitor, we show that the Erk/MAPK pathway is involved in IL-6-induced clonogenicity
of CD45+, but not CD45- myeloma cell lines. In contrast to IL-6, the other growth factors (IGF-1, FGF, HGF and
HB-EGF) stimulate only some myeloma cell lines, but always CD45-, and less effectively than IL-6. Among them,
IGF-1 is the most potent, generating myeloma colonies from five out of eight CD45- myeloma cell lines. Finally,
the capacity of IGF-1 and FGF to stimulate the clonogenicity of CD45- myeloma cells correlates with their ability
to stimulate the Erk/MAPK pathway. We conclude that CD45 expression plays a crucial role in determining
signaling and proliferation of human myeloma cell responses to IL-6, IGF-1 and other growth factors. The poor
outcome of CD45- myeloma patients could be related to the capacity of CD45-myeloma cells to take advantage of
multiple growth factors.
Keywords: multiple myeloma, clonogenicity, CD45IL-6, IGF-1
Multiple myeloma (MM) is a fatal, plasma cell malignancy
characterized by the accumulation of malignant plasma
cells within the bone marrow [1]. MM presents as a het-
erogeneous disease, with patients having very different
clinical outcomes. IL-6 and IGF-1 are known to be essen-
tial growth and survival factors in this malignancy [2-5].
IL-6 induces activation of both the Ras/MAP kinase and
the JAK/STAT pathways, the latter promoting MM cell
survival. On the other hand, activation of the IGF-1 recep-
tor (IGF-1R) results in activation of both the PI
3-kinase/Akt and the Ras/MAP kinase cascades. A signifi-
cant role for the PI 3-kinase/Akt pathway, as a mediator of
tumor expansion in MM, has been recently demonstrated
[6, 7]. Indeed, selective inhibition of the Akt pathway
results in both inhibition of MM cell proliferation [6] and
sensitization to apoptosis [7]. Furthermore, we have re-
cently provided evidence that the proliferation of myeloma
cells through the PI 3-kinase pathway, was clearly associ-
ated with the CD45- phenotype [8], which correlated to an
aggressive clinical presentation of MM [9], associated
with increased IGF-1 [8] and insulin receptor signaling
[10]. In addition to IL-6 and IGF-1, the heparin-binding
growth factors HGF, HB-EGF and FGF have all been
demonstrated to have a role in MM [11]. The heparan
sulfate syndecan (CD138), whose expression is a hallmark
of normal and malignant plasma cells, is able to bind
heparin-binding factors and to present them to their spe-
cific receptors. As with IGF-1, HGF, HB-EGF and FGF, all
activate both the PI 3-kinase/Akt and the Ras/MAP kinase
pathways [11] and, although all of them are involved in
myeloma cell proliferation, the relative importance of each
growth factor remains to be established. For this purpose,
we set up a myeloma cell colony–forming assay, which
does not allow the spontaneous formation of myeloma cell
colonies. This assay is highly efficient in comparing the
capacity of the different growth factors to stimulate the
generation of myeloma cell colonies. Thus, in the present
study, we investigated the capacity of IL-6, IGF-1, FGF,
HB-EGF and HGF to stimulate the generation of myeloma
cell colonies from fourteen, selected, human myeloma cell
lines (HMCL). The HMCL were carefully chosen to rep-
resent the phenotypic heterogeneity of MM and were seg-
regated into two groups based on CD45 expression.
MATERIALS AND METHODS
Human myeloma cell lines and culture conditions
LP-1, L363, NCI-H929 and OMP-2 HMCL were pur-
chased from DSM (Braunschweig, Germany) and RPMI-
8226 and U266 from the ATCC (Rockville, MA, USA).
JIM-3 and JJN-3 were kindly provided by Pr. L. Bergsagel,
1
The first two authors contributed equally to this work.
doi: 10.1684/ecn.2007.0095
Eur. Cytokine Netw., Vol. 18 n° 3, September 2007, 1-7 1
UNCORRECTED PROOF
USA and Pr. B. Van Camp, Belgium respectively. The
XG-1, XG-2, XG-6, NAN-1, NAN-4 and MDN HMCLs
had been previously established in our laboratory from
peripheral blood samples or pleural effusion of patients
with MM (see table) [12], and were cultured in the pres-
ence of 3 ng/ml of r-IL-6 (Novartis, Basel, Switzerland).
All HMCL expressed CD138 (table 1) [12]. Cell lines
were maintained in RPMI-1640 medium supplemented
with 10% FCS, 2 mM glutamine, antibiotics and 5x10
-5
M
2-bME.
Monoclonal antibodies (mAbs) and reagents
Human recombinant IGF-1 was purchased from Sigma (St
Louis, MI, USA). Human recombinant IL-6 was kindly
provided by Novartis. Human recombinant FGF, HB-EGF
and HGF were purchased from Preprotech (Rockhill, NJ,
USA). Anti-phospho -p44/42 MAP kinase, anti-p44/42
MAP kinase and anti-phospho-Akt (Ser 473) are from Cell
Signaling (Ozyme, Saint Quentin Yvelines, France).
U0126 and wortmannin are from Alexis Biochemicals
(Carlsbad, CA, USA).
Immunofluorescence analysis
Cells (0.5 x10
6
) were incubated with different PE-
conjugated mAb or anti-CD45-FITC (Beckman Coulter,
Marseilles) for 20 min at 4°C. The different PE-conjugated
mAb were anti-CD138, anti-CD126 from Beckman
Coulter, Marseilles, France and anti-CD38, anti-IGF-1R,
anti-CD45RA and anti-CD45RB from BD, Biosciences,
Le Pont de Claix, France. After two washes, cells were
fixed in 1% formaldehyde. Flow cytometry analysis was
performed on a FACSCalibur using the CELLQuest pro-
gram (Becton Dickinson, San Jose, CA, USA). The fluo-
rescence ratio was determined by dividing the mean fluo-
rescence intensity by the mean fluorescence intensity of
the respective control.
Myeloma cell colony-forming assay
Myeloma cells (10
3
cells) were plated in 1ml IMDM
serum-free, cytokine-free, human, purified collagen-
based, semi-solid medium (stema III, StemAlpha SA,
France) in triplicate (330ll/well), in 4-well plates and
grown for 15 days. For cytokine-stimulated assays in the
presence or not of inhibitors, cytokines and/or inhibitors
were mixed with the cell suspension in IMDM before
addition of the collagen. The gels were harvested on glass
slides, dried and stained with May-Grunwald-Giemsa.
Colonies were counted on triplicate gels by microscopy.
The number of colonies was expressed as an average per
10
3
cells.
Immunoblot analysis
Cells (4x10
6
) were resuspended in lysis buffer (10 mM
Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1 mM
PMSF, 2 mM Na
3
VO
4
, 1 mM NaF, 2 lg/mL aprotinin,
leupeptin 1lg/mL and 0.5% NP40). After 40 min on ice,
lysates were cleared by centrifugation at 12000xgfor
30 min at 4°C. Protein concentration was measured using
bicinchoninic acid (BCA protein assay, Pierce Rockford,
IL, USA). One hundred lg of proteins were loaded for
each lane. The proteins were separated by 10% SDS-
PAGE and then electrotransferred to PVDF membranes.
Western blot analysis was performed using standard tech-
niques with ECL detection (Roche, France).
Statistical analysis
The Fisher test was used for statistical analysis.
RESULTS
IL-6 is a clonogenic factor for both CD45+
and CD45- human myeloma cell lines (HMCL),
whereas IGF-1 and other growth factors (FGF, HGF
and HB-EGF) are clonogenic only for CD45- HMCL
This study was designed to compare the capacity of the
different myeloma cell growth factors to act as clonogenic
factors for HMCL in a collagen-based assay. The serum-
free and cytokine-free, collagen-based assay was designed
not to allow the generation of spontaneous myeloma colo-
Table 1
Characteristics of HMCL
HMCL Isotype Sample CD138 CD38 CD45 CD45RA CD45RO CD45RB CD126 CD221
LP1 IgG k PB ++----++
OPM2 IgG k PB ++----++
NCI-H929 IgA j PE + + 18% - 18% - + +
JIM-3 IgA PE + + - - - - + +
NAN-1 IgA j PE ++----++
RPMI8226 IgG k PB ++----++
JJN3 IgA j PE + + 43% - 43% - - +
L363 NS PE + + - - - - + +
XG-6 IgG k PB + - + + + + + +
NAN-4 IgA j PB + + + + + + + +
MDN IgG j PB + + + + - + + +
XG-1 IgA j PB + + + - + + + +
XG-2 IgG k PE + + + - + + + +
U266 IgE k PB + - 86% - 86% 86% + +
Expression of each molecule was evaluated by a single immunofluorescence assay. Positive expression of a molecule is defined by the level of intensity with a ratio > 2 (the ratio
is obtained by the ratio of fluorescence divided by the fluorescence of an isotypic control). -: not expressed, +: expressed as 100%, otherwise the% of positive cells is given. NS:
non-secreting, PB: peripheral blood, PE: pleural effusion.
2 M. Collette, et al.
UNCORRECTED PROOF
nies in the absence of exogenous growth factors, and, with
the exception of RPMI-8226 (10% of clonogenic cells)
and L363 (2% of clonogenic cells), no other HMCL was
able to generate significant numbers of colonies (>1% of
clonogenic cells) spontaneously. Fourteen HMCL were
studied, six expressed CD45 on a majority of cells (>80%),
and eight were lacking CD45 expression on a majority of
cells (> 50%). The observed CD45 expression corre-
sponded to the CD45RB isoform expression only (n = 1)
or the CD45RB isoform expression associated with either
CD45RA or RO (n = 3) or both CD45RA and CD45RO
(n = 2). IL-6 generated myeloma colonies in 10 out of the
14 HMCL. The clonogenicity ranged from 7% to 50%
(figure 1). Notably, IL-6 did not enhance the spontaneous
colony formation of RPMI-8226. In contrast to IL-6,
IGF-1 generated colonies in five out of the 14 HMCL,
inducing a weak effect (<3% of clonogenic cells) for L363
cells. This was not due to a lack of IGF-1R expression
since all 14 HMCL expressed the IGF-1R (table 1) [13].
The ability of FGF to stimulate colony formation was
restricted to LP1 and RPMI-8226 HMCL (figure 1). HGF
and HB-EGF had a very weak stimulating effect on colony
formation of LP1, RPMI-8226 and L363 (< 2% of clono-
genic cells) and of LP1 (3% of clonogenic cells), respec-
tively. Interestingly, the capacity of IGF-1, FGF, HGF and
HB-EGF to stimulate clonogenicity was restricted to
CD45- HMCL, whereas IL-6 was a clonogenic factor for
both CD45- and CD45+ HMCL. Finally, IGF-1 had a
broader range of activity than FGF, HGF and HB-EGF as it
was able to stimulate the clonogenicity in more HMCL.
Moreover, the number of colonies generated by IGF-1 was
greater compared to FGF, HGF or HB-EGF, except for
LP1, where FGF generated more (36% of clonogenic cells
with FGF versus 22% of clonogenic cells with IGF-1).
IL-6-induced colony formation involves the MAPK
pathway in CD45+ but not in CD45- HMCL
IL-6 triggers both the Ras/MAP kinase and the JAK/STAT
pathways, the latter promoting MM cell survival. Since,
IL-6 is a clonogenic factor for both CD45+ and CD45-
HMCL, we next examined whether the Erk pathway was
involved in clonogenicity, using the pharmacological
MEK1/2 inhibitor, U0126. U0126 inhibited the IL-6-
500
400
300
200
100
0
MCF /10
3
cells
Without cytokine IGF-1 (20 ng/ml)
LP1
OPM2
NCI-H929
JIM-3
JJN3
L363
XG-6
XG-1
XG-2
U266
NAN-4
MDN
NAN-1
RPMI-8226
IL-6 (20 ng/ml)
LP1
OPM2
NCI-H929
JIM-3
JJN3
L363
XG-6
XG-1
XG-2
U266
NAN-4
MDN
NAN-1
RPMI-8226
LP1
OPM2
NCI-H929
JIM-3
JJN3
L363
XG-6
XG-1
XG-2
U266
NAN-4
MDN
NAN-1
RPMI-8226
500
400
300
200
100
0
MCF /10
3
cells
FGF (50 ng/ml)
LP1
OPM2
NCI-H929
JIM-3
JJN3
L363
XG-6
XG-1
XG-2
U266
NAN-4
MDN
NAN-1
RPMI-8226
HB-EGF (1 µg/ml) HGF (200 ng/ml)
LP1
OPM2
NCI-H929
JIM-3
JJN3
L363
XG-6
XG-1
XG-2
U266
NAN-4
MDN
NAN-1
RPMI-8226
LP1
OPM2
NCI-H929
JIM-3
JJN3
L363
XG-6
XG-1
XG-2
U266
NAN-4
MDN
NAN-1
RPMI-8226
CD45+HMCLCD45-HMCL CD45+HMCLCD45-HMCL CD45+HMCLCD45-HMCL
Figure 1
Clonogenicity of CD45- and CD45+ HMCL. HMCL were separated into two groups according to their CD45 phenotype. CD45- HMCL (LP-1,
NCI-H929, JIM-3, NAN-1, RPMI-8226, JJN3, L363) and CD45+ HMCL (XG-6, NAN-4, MDN, XG-1, XG-2 and U266). CD45+ HMCL are
indicated in bold and italics. For myeloma colony assays, 10
3
myeloma cells were seeded per ml of serum-free, collagen-based, semi-solid stema
III medium with or without cytokines and grown for 15 days. Gels were then dried and stained with MGG, and myeloma colony formation (MCF)
was scored.
Identification of clonogenic factors for myeloma cells 3
UNCORRECTED PROOF
induced clonogenicity of all CD45+ HMCL tested with an
inhibition ranging from 36% to 67% (mean value
m = 53%) (figure 2). In contrast, U0126 only reduced the
clonogenicity (16%) of one out of five CD45- HMCL
tested. Of note, U0126 strongly increased the IL-6-
induced clonogenicity of NCI-H929 (243% increase) (fig-
ure 2)). Altogether, these results demonstrated that the
Erk/MAPK cascade was significantly involved in IL-6-
induced clonogenicity in CD45+HMCL but not CD45-
HMCL (p < 0.05) (Fisher test).
Significant Erk phosphorylation induction in response
to IGF-1 or FGF correlates with the capacity
to generate colony formation
We searched for a correlation between clonogenicity and
the signaling pathway activated by IGF-1 and FGF in MM
cells. We and others, have recently demonstrated that PI-3
kinase pathway activation was induced in all HMCL in
response to IGF-1. However, the magnitude of Akt phos-
phorylation in response to IGF-1 was greater in CD45-
than in CD45+ HMCL [8]. Finally, the strong activation of
the PI 3-kinase pathway in all CD45- HMCL does not
seem to be sufficient to explain the IGF-1-restricted clono-
genicity of five out out the eight CD45- HMCL. Thus, we
focused on Erk phosphorylation induced by IGF-1 and
FGF. Under IGF-1 stimulation, Erk phosphorylation was
induced in L363, JIM-3, LP-1 and RPMI-8226 CD45-
HMCL (figure 3A), whereas induction of Erk phosphory-
lation was undetectable in NAN-4, XG-1, XG-6 and MDN
CD45+ HMCL. A kinetic study of Erk phosphorylation in
a CD45-HMCL (L363) indicated that the ERK response
was similar between 15 min to 120 min under IGF-1
stimulation (figure 3B). Similar kinetics in a CD45+
HMCL (NAN-4) confirmed that the absence of ERK in-
duction of CD45+ HMCL was not due to a difference in
kinetic response, but to a total absence of response. Alto-
gether, these results demonstrated that the ERK response
to IGF-1 is significantly different between CD45- HMCL
and CD45+ HMCL (p < 0.05). Of interest, the ERK phos-
phorylation induced by IGF-1 was completely restricted to
the CD45- HMCL able to generate myeloma colonies in
the presence of IGF-1. The same analysis was performed
with FGF, demonstrating that induction of ERK phospho-
rylation by FGF is restricted to LP1 and RPMI-8226 (fig-
ure 3C). Thus, the activation of the MAPK pathway by
IGF-1 or FGF correlated with the capacity of these factors
to generate myeloma colonies. Taken together, these re-
sults highlight the importance of the ERK/MAPK pathway
in IGF-1- or FGF-induced clonogenicity of CD45- HMCL.
IL-6 and growth factors do not co-operate to induce
clonogenicity, whereas IGF-1 and HGF can synergize
to induce colony formation
Since cross-talk between IL-6R and IGF-1R has been
demonstrated [14], we next evaluated the potential of
IGF-1 and IL-6 in combination, to generate myeloma cell
colonies. In all HMCL tested (n = 5), the number of my-
eloma cell colonies induced by IGF-1 and IL-6 combina-
tion was never higher than that induced by IL-6 alone,
indicating that IL-6 and IGF-1 do not co-operate in colony
formation induction (figure 4). Similarly, we found that the
combination of IL-6 with the other growth factors (FGF,
HB-EGF and HGF), did not co-operate to induce clonoge-
nicity (result not shown). Consistent with these results, in
CD45- HMCL, where both cytokines induced the
Erk/MAPK pathway, the combination of IL-6 and IGF-1
did not result in an additive effect of Erk phosphorylation
(figure 5B). Finally, we analyzed the effect of IGF-1 in
combination with the other growth factors (FGF, HB-EGF
and HGF); we found that the combination of IGF-1 and
400
500
600
300
200
100
0
MCF/10
3
cells
LP1
OPM2
JIM-3
L363
NAN-4
MDN
XG-1
XG-2
NCI-H929
CD45-HMCL CD45+HMCL
RPMI-8226
IL-6 (20 ng/ml)
IL-6 (20 ng/ml)
+ U0126 (10 µM)
Figure 2
The Erk/MAPK pathway is involved in IL-6-induced clonogenicity of CD45+ HMCL only. Myeloma cells (10
3
) were seeded per ml of
serum-free, collagen-based, semi-solid stema III medium containing IL-6, with or without inhibitor (U0126) and grown for 15 days. Gels were
then dried and stained with MGG, and MCF was scored. Values represent the mean ± SD of three experiments of duplicate cultures.
4 M. Collette, et al.
UNCORRECTED PROOF
HGF synergized in inducing clonogenicity. As illustrated
in figure 5A, a marked increase in clonogenicity was
observed with the combination of IGF-1 and HGF com-
pared to IGF-1 alone (324 ±1 colonies versus 134±4 colo-
nies) for NCI-H929. Consistent with this, IGF-1 in combi-
nation with HGF, and compared to IGF-1 alone, was
associated with a marked increase in the levels of both Erk
phosphorylation (324% increase) andAkt phosphorylation
(133% increase) (figure 5B).
DISCUSSION
In the present study, we have described a serum-free,
cytokine-free, collagen-based assay that identified the ca-
pacity of an isolated cell to self-renew only when the right
growth factor or combination of growth factors were
present. This assay also allowed the prioritization of spe-
cific clonogenic factors for HMCL. Furthermore, we uti-
lized a panel of heterogeneous HMCL reflecting the ge-
netic diversity of clinical MM. With the exception of
RPMI-8226, none of the HMCL retains the capacity to
self-renew and proliferate in the absence of cytokines or
growth factors. We identified IL-6 as a ubiquitous clono-
genic factor for human MM cells that acted independently
of their CD45 phenotype. Of note, all HMCL expressed
A
B
C
JIM-3
IGF-1 100 ng/ml - - - - - ++++++-
p-Erk
Erk
IGF-1 100 ng/ml 0 15’ 30’ 60’ 30’ 60’ 120’120’ 0
p-Erk
Erk
FGF 50 ng/ml
p-Erk
Erk
LP-1
L363
8226
NAN-4
RPMI-
MDN
RPMI-
8226
-+ -+-+-+
NCI-
H929
LP-1
XG-1 XG-6 MDN
Figure 3
Analysis of ERK phosphorylation induced by IGF-1 or FGF in HMCL. Eighteen- hour, serum-starved cells were treated or not with IGF-1 for
the indicated time (figure 5B) or with IGF-1 or FGF for 30 minutes (figure 5A and 5C). Equivalent amounts of cell lysates were separated by
SDS-PAGE, then immunoblotted with anti-phospho-ERK antibodies. Protein loading was controlled with an anti-ERK total “total”: OK ?.
600
500
400
300
200
100
0
LP-1 L363
IL-6
IGF-1
IL-6+IGF-1
CD45-HMCL
MCF/10
3
cells
CD45+HMCL
XG-2 XG-6NCI-
H929
Figure 4
Effect of the combination of IL-6 +IGF-1 on colony formation.
Myeloma cells (10
3
) were seeded per ml of serum-free, collagen
–based, semi-solid stema III medium containing IL-6, IGF-1 or
IL-6 + IGF-1 and grown for 15 days. Gels were then dried and
stained with MGG, and colony formation was scored. Values repre-
sent the mean ± SD of three experiments of duplicate cultures.
Identification of clonogenic factors for myeloma cells 5
UNCORRECTED PROOF
the IL-6R except JJN-3, which is one of the two HMCL
unable to clone under the influence of IL-6. These data are
consistent with the major role of IL-6 in the proliferation
and survival of myeloma cells [11, 15]. The level of clono-
genicity induced by IL-6 of up to 50%, clearly indicated
that clonogenic cells in HMCL are highly representative.
IL-6 triggers both the Ras/MAP kinase and the JAK/STAT
pathways, the latter promoting MM cell survival [16].
Thus, we investigated the involvement of Ras/MAPK
pathway using a specific MEK 1/2 inhibitor, U0126. Our
results demonstrated that this pathway is involved in IL-6-
induced colony formation of CD45+ HMCL but not in that
of CD45-HMCL. Consistent with these results, previous
data have shown that activation of src kinase is also depen-
dent on CD45 expression and is necessary to induce IL-6-
dependent proliferation [17, 18]. Moreover, translocation
of CD45 to lipid rafts is also required to confer the ability
to respond to IL-6. This is consistent with our observation
that all the CD45+ HMCL expressed the CD45RB isoform
of CD45, which, unlike CD45RA, is able to translocate to
lipid rafts [19]. Thus, both the Ras/MAP kinase pathway
and src activation are necessary for the IL-6-induced pro-
liferation and clonogenicity of CD45+ HMCL, whereas
IL-6 is a clonogenic factor for CD45- HMCL, independent
of these pathways. Further investigations are necessary to
elucidate the signaling pathway responsible for the latter
observation. In this respect, a working hypothesis will be
that IL-6 induces the generation of an autocrine clonogenic
factor in CD45- HMCL. Although this factor has not yet
been identified, we can already exclude the growth factors
tested in this study. Furthermore, the inability of IGF-1
inhibition to prevent IL-6-induced clonogenicity, and the
ability of Baff to induce colony formation in only a limited
number of HMCL (LP1, L363 and RPMI-8226) (data not
shown) would argue against a role for an autocrine produc-
tion of these factors in IL-6-induced clonogenicity. Inter-
estingly, our study demonstrated that whereas CD45+
HMCL could benefit from IL-6 only, CD45- myeloma
cells responded to both IL-6 and a range of other growth
factors (IGF-1, FGF, HB-EGF, HGF) to generate myeloma
colonies. The capacity of IGF-1 to generate myeloma
colonies correlated with the induction of Erk /MAPK
signaling and activation of the PI 3-kinase pathway. In-
deed, IGF-1 was unable to activate the Erk/MAPK path-
way or induce colony formation in CD45+ HMCL. More-
over, in the CD45-HMCL, IGF-1 synergized with HGF to
induce colony formation, as well as Erk/MAPK and Akt
activation. Finally, it has been previously reported that
IL-6 and growth factors co-operate to induce MM cell
growth [13, 20, 21]. However, in our semi-solid assay, we
observed that IL-6 did not synergize with IGF-1, FGF,
HB-EGF or HGF, and indeed the combination of IL-6 and
IGF-1 was inhibitory. In accordance with these data, in cell
lines where IGF-1 or IL-6 induced Erk activation, this
activation was never enhanced with IL-6 and IGF-1 com-
binations. A more detailed analysis of cross-talk between
these factors will help us to understand these results.
In conclusion, CD45 is essential for the control of signal-
ing and proliferation of human myeloma cells in response
to IL-6, IGF-1 and other growth factors. The poor outcome
of CD45-myeloma patients could be related to the capacity
of CD45- myeloma cells to take advantage of multiple
growth factors. Thus, treatment strategies for CD45- pa-
tients should combined the disruption of signaling induced
by both IL-6 and IGF-1, and other growth factors.
Acknowledgments. We thank Dr Andrew Spencer for the criti-
cal reading of the manuscript.. This work was supported by
The Ligue Nationale Contre le Cancer (équipe labelisée 2005).
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0
MCF/10
3
cells
A
B
NCI-H929
NCI-H929
IL-6 50 ng/ml
-+ -++-
-- --++
--++--
IGF-1 100 ng/ml
HGF 200 ng/ml
p-Ser473 Akt
p-Erk-1/2
Erk-1/2
IL-6
IGF-1
IL-6+IGF-1
HGF
HGF+IGF-1
Figure 5
Synergistic effect of the combination of IGF-1+HGF on NCI-H929
colony formation. A) Myeloma cells (10
3
) were seeded per ml of
serum-free, collagen-based, semi-solid stema III medium containing
the indicated growth factor and grown for 15 days. Gels were then
dried and stained with MGG, and MCF was scored. Values represent
the mean ± SD of three experiments of duplicate cultures. B) The
synergistic effect of the combination of IGF-1+HGF on colony
formation is mediated by a stronger activation of both PI-3K/Akt and
Erk/MAPK pathways. Eighteen-hour, serum-starved cells for were
treated or not with IGF-1, IL-6, IGF-1+IL-6, HGF or IGF-1+HGF for
30 minutes. Equivalent amounts of cell lysates were separated by
SDS-PAGE, then immunoblotted with anti-phospho-ERK or anti-
phospho Akt antibodies. Protein loading was controlled with an
anti-ERK total “total”: OK ?.
6 M. Collette, et al.
UNCORRECTED PROOF
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Identification of clonogenic factors for myeloma cells 7
... Finally, PTPRC and CDH5 have not been previously reported to be associated with cervical cancer or its lymph node metastasis. While experiments demonstrate that PTPRC expression is increased in other cancers and their lymph node metastases (Collette et al., 2007;Camacho et al., 2018), CDH5 is up-regulated in other cancers but down-regulated in lymph node metastasis of colorectal cancer (Tacconi et al., 2015;Hung et al., 2016;Higuchi et al., 2017). ...
... Camacho et al. (2018) showed that PTPRC is significantly overexpressed in head and neck squamous cell cancer cells, and tumor samples overexpressing PTPRC have significantly higher tumor-infiltrating lymphocytes (TIL) scores than tumor samples expressing low levels of PTPRC, leading to a poorer prognosis. Collette et al. (2007) noted that the expression of PTPRC plays a critical role in determining the signal transduction and proliferation response of human myeloma cells to growth factors such as IL-6 and IGF1. IL-6 and IGF1 separately induced CD45+ and CD45-myeloma cell colony formation through the MAPK/ERK signaling pathway in which CD45 is critical for myeloma proliferation (Collette et al., 2007). ...
... Collette et al. (2007) noted that the expression of PTPRC plays a critical role in determining the signal transduction and proliferation response of human myeloma cells to growth factors such as IL-6 and IGF1. IL-6 and IGF1 separately induced CD45+ and CD45-myeloma cell colony formation through the MAPK/ERK signaling pathway in which CD45 is critical for myeloma proliferation (Collette et al., 2007). In addition, many experimental studies reported that the MAPK/ERK signaling pathway is crucial in cervical cancer formation. ...
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Background: miRNAs and genes can serve as biomarkers for the prognosis and therapy of cervical tumors whose metastasis into lymph nodes is closely associated with disease progression and poor prognosis. Methods: R software and Bioconductor packages were employed to identify differentially expressed miRNAs (DEMs) from The Cancer Genome Atlas (TCGA) database. GEO2R detected differentially expressed genes (DEGs) in the GSE7410 dataset originating from the Gene Expression Omnibus (GEO). A Cox proportional hazard regression model was established to select prognostic miRNA biomarkers. Online tools such as TargetScan and miRDB predicted target genes, and overlapping DEGs and target genes were defined as consensus genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) function annotations were performed to discern the potential functions of consensus genes. STRING and Cytoscape screened key genes and constructed a regulatory network. Results: A combination of four miRNAs (down-regulated miR-502 and miR-145, up-regulated miR-142 and miR-33b) was identified as an independent prognostic signature of cervical cancer. A total of 94 consensus genes were significantly enriched in 7 KEGG pathways and 19 GO function annotations including the cAMP signaling pathway, the plasma membrane, integral components of the plasma membrane, cell adhesion, etc. The module analysis suggested that CXCL12, IGF1, PTPRC CDH5, RAD51B, REV3L, and WDHD1 are key genes that significantly correlate with cervical cancer lymph node metastasis. Conclusions: This study demonstrates that a four-miRNA signature can be a prognostic biomarker, and seven key genes are significantly associated with lymph node metastasis in cervical cancer patients. These miRNAs and key genes have the potential to be therapeutic targets for cervical cancer. Among them, two miRNAs (miR-502 and miR-33b) and two key genes (PTPRC and CDH5) were first reported to be potential novel biomarkers for cervical cancer. The current study further characterizes the progression of lymph node metastasis and mechanism of cervical tumors; therefore, it provides a novel diagnostic indicator and therapeutic targets for future clinical treatments.
... This finding supports the notion that CD45 + immature myeloma cells are growing but are mortal, depending on the environment, whereas CD45cells are resting but relatively resistant to apoptosis [22]. Moreover, even though IL-6 is a growth factor only for CD45 + human MM cell lines (HMCLs), other growth factors such as insulin-like growth factor 1 (IGF1), fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are growth promoting only for CD45 -HMCLs [30]. It should be noted that the amount of IL-6 is limited in the BM of MM patients [22]. ...
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Many cell signaling pathways are activated or deactivated by protein tyrosine phosphorylation and dephosphorylation, catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. Even though PTPs are as important as PTKs in this process, their role has been neglected for a long time. Multiple myeloma (MM) is a cancer of plasma cells, which is characterized by production of monoclonal immunoglobulin, anemia and destruction of bone. MM is still incurable with high relapse frequency after treatment. In this review, we highlight the PTPs that were previously described in MM or have a role that can be relevant in a myeloma context. Our purpose is to show that despite the importance of PTPs in MM pathogenesis, many unanswered questions in this field need to be addressed. This might help to detect novel treatment strategies for MM patients.
... IL-6 is a pleotropic cytokine with effects on numerous target cells through activation of the antiapoptotic Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway [63,64]. IL-6 is a powerful myeloma cell growth factor -within the bone marrow, IL-6 produced by both stromal or MM cells has been found to promote myeloma cell proliferation and survival [61][62][63][64][65]. IL-6 and its surrogate markers are poor prognostic factors in MM [66]. ...
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Multiple myeloma (MM) is a haematological malignancy arising from monoclonal proliferation of plasma cells in the bone marrow, resulting in the presence of paraproteins or M-protein in serum. The involvement of paraproteins produced by malignant plasma cells in the development of hyperlipidaemia and low-HDL cholesterol has been described, as has an association with MM and obesity, hypertension, and type 2 diabetes mellitus, and insulin resistance, that is, features of the metabolic syndrome (MS). There is an association between MS components, inflammatory cytokines, and the development of MM, and some drugs used in the treatment of MS such as statins and metformin may improve outcomes in MM.
... en, we constructed the PPI network to better understand the interactions of DEGs, and the top three modules show that they were all significantly related to the pathway of immunologic and inflammatory response. CCR7, PTPRC (CD45), and IL10 were the most connected nodes in these modules, in which CCR7 was a crucial molecule in the mechanism of HCC's progression and metastasis [27][28][29], PTPRC involved in the regulation of cytokine-induced signaling in malignancies [30][31][32], and IL10 has been reported to increase the susceptible risk of HCC [33], decrease immunologic activity [34], and promote immune tolerance in the tumor milieu [35,36]. Finally, survival analysis was performed to explore the potential prognostic value of 289 DEGs, and we identified 12 TME-related genes that showed significant correlation between gene expression and poor outcomes in HCC cases. ...
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Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies. Recent studies reveal that tumor microenvironment (TME) components significantly affect HCC growth and progression, particularly the infiltrating stromal and immune cells. Thus, mining of TME-related biomarkers is crucial to improve the survival of patients with HCC. Public access of The Cancer Genome Atlas (TCGA) database allows convenient performance of gene expression-based analysis of big data, which contributes to the exploration of potential association between genes and prognosis of a variety of malignancies, including HCC. The “Estimation of STromal and Immune cells in MAlignant Tumors using Expression data” algorithm renders the quantification of the stromal and immune components in TME possible by calculating the stromal and immune scores. Differentially expressed genes (DEGs) were screened by dividing the HCC cohort of TCGA database into high- and low-score groups according to stromal and immune scores. Further analyses of functional enrichment and protein-protein interaction networks show that the DEGs are mainly involved in immune response, cell adhesion, and extracellular matrix. Finally, seven DEGs have significant association with HCC poor outcomes. These genes contain FABP3 , GALNT5 , GPR84 , ITGB6 , MYEOV , PLEKHS1 , and STRA6 and may be candidate biomarkers for HCC prognosis.
... This intra-clonal phenotypic heterogeneity is most often seen for the expression of CD45, a phosphatase which regulates the effects of MM cell growth factors on MM cells, actually which regulates the availability of the major growth factors IL6 and IGF1 (as nutrients) for MM subclones. This regulation operates through the capacity of CD45 to facilitate either SRC kinase activity (IL6 signaling) when present at the surface of MM cells, or RTK activity (IGF1R/CD221 and IGF1 signaling) when absent since CD45 is a potent inhibitor of IGF1R (CD221) aberrantly expressed on MM cells (42,43). ...
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The standard model of multiple myeloma (MM) oncogenesis is based on the genetic instability of MM cells and presents its evolution as the emergence of clones with more and more aggressive genotypes, giving them surviving and proliferating advantage. The micro-environment has a passive role. In contrast, many works have shown that the progression of MM is also characterized by the selection of clones with extended phenotypes able to destroy bone trabeculae, suggesting a major role for early micro-environmental disruption. We present a model of MM oncogenesis in which genetic instability is the consequence of the disruption of normal interactions between plasma cells and their environment, the bone remodeling compartment. These interactions, which normally ensure the stability of the genotypes and phenotypes of normal plasma cells could be disrupted by many factors as soon as the early steps of the disease (MGUS, pre-MGUS states). Therapeutical implications of the model are presented.
... 7,17 In summary, PTPRC is essential for the normal development of immune cells and their effector functions, acting as a regulator of a variety of cellular processes, and can be involved in the mechanism of cell survival, 18 apoptosis, 10 and tumor progression. 19 ...
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The protein tyrosine phosphatase receptor type C (PTPRC), also known as CD45 molecule, is a glycoprotein expressed on the cell surface of all hematopoietic cells except erythrocytes. Functionally, PTPRC is an immunomodulatory gene required for the efficient development of the immune system where it is involved in antigen receptor signal transduction. Several single nucleotide polymorphisms of the PTPRC gene have been described. In humans, the most extensively reported PTPRC polymorphism is the C77G point mutation in exon 4, and this nucleotide transversion causes abnormal PTPRC splicing, thus resulting in an altered expression pattern of isoforms with enhanced expression of high-molecular-weight isoforms (CD45RA, CD45RB, and CD45RC) and decreased low-molecular-weight CD45RO molecules. This altered expression pattern of isoforms can have significant effects on immune function, autoimmunity, and viral infections. Previous epidemiological studies have investigated the relationship between the C77G variant and several diseases. An association between this polymorphism and susceptibility to multiple sclerosis (MS) has been reported in some research papers, thereby suggesting a possible etiologic role of PTPRC in the development of this disease. Subsequent studies performed in other populations, including family-based and case-control studies, could not replicate this relationship between MS and the C77G point mutation, thus suggesting that more statistically powered studies are needed to confirm such an association or not. Furthermore, C77G polymorphism has been suggested to contribute to the development of some infective or autoimmune disorders. As individuals with C77G polymorphism may have increased susceptibility to HIV-1 infection, the frequency of this variant has been investigated in hepatitis C, histiocytosis, and autoimmune diseases, with contrasting results. Although PTPRC represents one of the modifier genes of human autoimmunity, further studies are needed to explain the exact role of PTPRC gene C77G variant in the contribution to the alteration of immune responses in infectious and autoimmune diseases.
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soutenance le 30 novembre 2009
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Thesis
Cancer cells frequently escape from immune surveillance by down-regulating two important components of the immune defence: antigen-presenting MHC and costimulatory molecules. Therefore several novel anti-tumour compounds that aim to assist the immune system in recognising and fighting cancer are currently under development. Recombinant bispecific antibodies represent one group of such novel therapeutics. They target two different antigens and recruit cytotoxic effector cells to tumour cells. For cancer immunotherapy, bispecific T cell-engaging antibodies are already well characterised. These antibodies target a tumour-associated antigen and CD3ε, the constant molecule of the T cell receptor complex. On the one hand, this study presents the development of a bispecific antibody targeting CD3ε and the rhabdomyosarcoma-associated fetal acetylcholine receptor. On the other hand, it describes a novel two-part trispecific antibody format for the treatment of leukaemia and other haematological malignancies in the context of haematopoietic stem cell transplantation (HSCT). For HSCT, an HLA-identical donor is preferred, but very rarely available. In an HLA-mismatched setting, the HLA disparity could be exploited for targeted cancer treatment. In the present study, a two-part trispecific HLA-A2 × CD45 × CD3 antibody was developed for potential cases in which the patient is HLA-A2-positive, but the donor is not. This holds true for about half the cases in Germany, since HLA-A2 is the most common HLA molecule found here. Combinatorial targeting of HLA-A2 and the leucocyte-common antigen CD45 allows for highly specific dual-antigen restricted tumour targeting. More precisely, two single-chain antibody constructs were developed: i) a single-chain variable fragment (scFv) specific for HLA-A2, and ii) a scFv against CD45, both linked to the VL and the VH domain of a CD3ε-specific antibody, respectively. It turned out that, after the concomitant binding of these constructs to the same HLA-A2- and CD45-expressing cell, the unpaired variable domains of a CD3ε-specific antibody assembled to a functional scFv. In a therapeutic situation, this assembly should exclusively occur on the recipient’s blood cancer cells, leading to T cell-mediated cancer cell destruction. In this way, a relapse of disease might be prevented, and standard therapy (radiation and chemotherapy) might be omitted. For both approaches, the antibody constructs were periplasmically expressed in E. coli, purified via His tag, and biochemically characterised. Their binding to the respective targets was proven by flow cytometry. The stimulatory properties of the antibodies were assayed by measuring IL-2 release after incubation with T cells and antigen-expressing target cells. Both the bispecific antibody against rhabdomyosarcoma and the assembled trispecific antibody against blood cancer mediated T-cell activation in a concentration-dependent manner at nanomolar concentrations. For the trispecific antibody, this effect indeed proved to be dual antigen-restricted, as it could be blocked by prior incubation of either HLA-A2- or CD45-specific scFv and did not occur on single-positive (CD45+) or double-negative (HLA-A2- CD45-) target cells. Furthermore, antibodies from both approaches recruited T cells for tumour cell destruction in vitro.
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Human B cell stimulatory factor 2 (BSF-2) was originally characterized and isolated as a T cell-derived factor that caused the terminal maturation of activated B cells to immunoglobulin-producing cells. Molecular cloning of the complementary DNA predicts that BSF-2 is a protein of relative molecular mass (Mr) 26,000 similar or identical to interferon beta 2, hybridoma plasmacytoma growth factor and hepatocyte stimulating factor. IL-6 has been proposed as a name for this molecule. It is now known that BSF-2 has a wide variety of biological functions and that its target cells are not restricted to normal B cells. Responses are also seen in T cells, plasmacytomas, hepatocytes, haematopoietic stem cells, fibroblasts and rat phoeochromocytoma, PC12 (Satoh, T. et al., manuscript in preparation). Of particular interest to this report is that human BSF-2 is a potent growth factor for murine plasmacytomas and hybridomas. This observation suggested to us that constitutive expression of BSF-2 or its receptor could be responsible for the generation of human myelomas. In this study we report that myeloma cells freshly isolated from patients produce BSF-2 and express its receptors. Moreover, anti-BSF-2 antibody inhibits the in vitro growth of myeloma cells. This is direct evidence that an autocrine loop is operating in oncogenesis of human myelomas.
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Human multiple myeloma (MM) represents a highly aneuploid tumor as shown by cytogenetic studies. This may partly explain the heterogeneity with regard to growth factor requirements demonstrated among MM cells. We have previously reported the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNA in some MM cell lines. In this study we investigated the role of IGF-I as a growth and/or survival factor in three MM cell lines: LP-1, EJM, and Karpas 707. We report that all cell lines expressed IGF-I and IGF-IR mRNA and protein. LP-1 and Karpas 707, but not EJM, were stimulated to proliferation in a dose-dependent manner by exogenous IGF-I. An IGF-IR blocking antibody inhibited both the IGF-I-induced and spontaneous growth of LP-1, and Karpas 707, while the EJM cell line was unaffected by the addition of the antibody. In conclusion, our results show that IGF-I can act as a growth factor in human MM, and they suggest that an autocrine IGF-I loop may contribute to the growth and survival in some MM cell lines.
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Multiple myeloma (MM) is an invariably fatal disease that accounts for approximately 1% to 2% of all human cancers. Surprisingly little is known about the cellular pathways contributing to growth of these tumors. Although the cytokine interleukin-6 has been suggested to be the major stimulus for myeloma cell growth, the role of a second potential growth factor, insulin-like growth factor I (IGF-I), has been less clearly defined. The IGF-I signaling cascade in 8 MM cell lines was examined. In 7 of these, the IGF-I receptor (IGF-IR) was expressed and autophosphorylated in response to ligand. Downstream of IGF-IR, insulin receptor substrate 1 was phosphorylated, leading to the activation of phosphatidylinositol-3'-kinase (PI-3K). PI-3K, in turn, regulated 2 distinct pathways. The first included Akt and Bad, leading to an inhibition of apoptosis; the second included the mitogen-activated protein kinase (MAPK), resulting in proliferation. Biologic relevance of this pathway was demonstrated because in vitro IGF-I induced both an antiapoptotic and a proliferative effect. Importantly, in vivo administration of IGF-I in SCID mice inoculated with the OPM-2 line led to approximately twice the growth rate of tumor cells as in controls. These results suggest that IGF-I activates at least 2 pathways effecting myeloma cell growth and contributes significantly to expansion of these cells in vivo. (Blood. 2000;96:2856-2861)
Article
Interleukin 6 (IL-6) and insulin-like growth factor I (IGF-I) induce proliferative and antiapoptotic responses in multiple myeloma (MM) plasma cells. Because these cytokines may activate the phosphatidylinositol 3-kinase (PI 3-K)/AKT kinase pathway in other cell types, we investigated the role of PI 3-K/AKT in MM cell responses. IGF-I effectively activated PI 3-K in 8226 and OCI-My5 MM cells, but IL-6 was ineffective. However, IL-6 successfully activated PI 3-K in AF-10 MM cells and IL-6-dependent MH.60 plasmacytoma/hybridoma cells. IGF-I also successfully activated PI 3-K in four of four MM patient specimens, and IL-6 activated PI 3-K in three of four specimens. Inhibition of PI 3-K activity with wortmannin or Ly294002 blocked the antiapoptotic effect of IGF-I and the proliferative effect of IL-6 in the myeloma cell lines. Furthermore, a dominant negative PI 3-K construct, expressed in AF-10 cells by adenoviral infection, also significantly inhibited the IL-6 proliferative response in MM cells. In correlation with activation of PI 3-K, IGF-I also effectively activated the AKT kinase in 8226 and OCI-My5 cells, and IL-6 activated AKT in AF-10 and MH.60 cells. However, although incapable of activating PI 3-K in 8226 and OCI-My5 cells, IL-6 successfully activated AKT in these MM lines, suggesting PI 3-K-independent mechanisms of AKT activation. The prevention of a myeloma cell proliferative response resulting from inhibition of PI 3-K activity was not associated with an inhibition of IL-6-dependent extracellular signal-regulated kinase (ERK) activation. These results support a role for the PI 3-K/AKT pathway in cytokine-dependent responses in myeloma cells, which is independent of any activation of the ERK pathway.
Article
Multiple myeloma (MM) is a plasma-cell disorder in which malignant plasma cells accumulate in the bone marrow and usually produce a monoclonal immunoglobulin. Usual presenting features of overt MM include recurrent osteolytic lesions, bacterial infections, anemia and renal insufficiency. MM is responsible for about 1 percent of all cancer-related deaths in Western countries. Its epidemiologic pattern remains obscure, and its cause unknown [1]. The presence of somatic mutations within the immunoglobulin genes of myeloma cells indicate that the putative myeloma-cell precursors have been stimulated by antigens within germinal centers and are either memory B cells or migrating plasmablasts. Myeloma cells proliferate slowly in the bone marrow and display a weak apoptotic index in vivo [2]. This suggest that some defects in the apoptotic process could be involved in this neoplasia. Interleukin-6 (IL-6) is known to be an essential survival factor of myeloma cells and to protect them from apoptosis induced by different stimuli (e.g. dexamethasone, CD95, serum starvation, gamma-irradiation). More recently, important works have been devoted to the biology of the soluble form of the IL-6R alpha i.e., sIL-6R alpha. These works give IL-6/sIL-6R alpha complex an important role in the biology of IL-6. The purpose of the current review is to emphasize the role of this complex in the pathogenesis of MM.
Article
Previous studies demonstrate that interleukin-6 (IL-6) mediates growth and survival in human multiple myeloma (MM) cells via the MEK/MAPK and JAK/STAT signaling pathways, respectively. IL-6 also confers protection against Dexamethasone (Dex)-induced apoptosis via activation of protein tyrosine phosphatase (SHP2). In the current study, we characterized IL-6 triggered phophatidylinositol-3 kinase/Akt kinase (PI3-K/Akt) signaling in MM cells. IL-6 induces Akt/PKB phosphorylation in a time and dose dependent manner in MM.1S MM cells. IL-6 also induced phosphorylation of downstream targets of Akt, including Bad, GSK-3beta, and FKHR, confirming Akt activation. Inhibition of Akt activation by the PI3-K inhibitor LY294002 partially blocked IL-6 triggered MEK/MAPK activation and proliferation in MM.1S cells, suggesting cross-talk between PI3-K and MEK signaling. We demonstrate that Dex-induced apoptosis in MM.1S cells is mediated by downstream activation of caspase-9, with resultant caspase-3 cleavage; and conversely, that IL-6 triggers activation of PI3-K and its association with SHP2, inactivates caspase-9, and protects against Dex-induced apoptosis. LY294002 completely abrogates this signaling cascade, further confirming the importance of PI3-K/Akt signaling in conferring the protective effect of IL-6 against Dex-induced apoptosis. Finally, we show that IL-6 triggered PI3-K/Akt signaling in MM.1S cells inactivates forkhead transcriptional factor (FKHR), with related G1/S phase transition, whereas LY294002 blocks this signaling, resulting in upregulation of p27(KIP1) and G1 growth arrest. Our data therefore suggest that PI3-K/Akt signaling mediates growth, survival, and cell cycle regulatory effects of IL-6 in MM.