Oncogene (2011) 30, 3198–3206
& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11
www.nature.com/onc
SHORT COMMUNICATION
Serum/glucocorticoid-regulated kinase 1 (SGK1) is a prominent target
gene of the transcriptional response to cytokines in multiple myeloma
and supports the growth of myeloma cells
U-M Fagerli1,2, K Ullrich3,4, T Stühmer5, T Holien1, K Köchert3,4, RU Holt1,6, O Bruland7,
M Chatterjee5, H Nogai3, G Lenz3, JD Shaughnessy Jr8, S Mathas3,4, A Sundan1, RC Bargou5,
B Dörken3,4, M Brset1,9 and M Janz3,4
1
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway;
Department of Oncology, St Olavs University Hospital, Trondheim, Norway; 3Department of Hematology, Oncology and
Tumorimmunology, Charité, University Medical School Berlin, Berlin, Germany; 4Hematology, Oncology and Tumorimmunology,
Max Delbrück Center for Molecular Medicine, Berlin, Germany; 5Department of Internal Medicine II and Comprehensive Cancer
Center Mainfranken, Division of Hematology, University Hospital Würzburg, Würzburg, Germany; 6Department of Biomedical
Science, Faculty of Technology, Sr-Trndelag University College, Trondheim, Norway; 7Center of Medical Genetics and Molecular
Medicine, Haukeland University Hospital Bergen, Bergen, Norway; 8Myeloma Institute for Research and Therapy, University
of Arkansas for Medical Sciences (UAMS), Little Rock, AR, USA and 9Department of Immunology and Transfusion Medicine,
St Olavs University Hospital, Trondheim, Norway
2
Multiple myeloma (MM) is a paradigm for a malignant
disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding
of the complex interactions between malignant plasma
cells and their surrounding requires a detailed analysis of
the transcriptional response of myeloma cells to environmental signals. We determined the changes in gene
expression induced by interleukin (IL)-6, tumor necrosis
factor-a, IL-21 or co-culture with bone marrow stromal
cells in myeloma cell lines. Among a limited set of genes
that were consistently activated in response to growth
factors, a prominent transcriptional target of cytokineinduced signaling in myeloma cells was the gene encoding
the serine/threonine kinase serum/glucocorticoid-regulated kinase 1 (SGK1), which is a down-stream effector
of PI3-kinase. We could demonstrate a rapid, strong and
sustained induction of SGK1 in the cell lines INA-6,
ANBL-6, IH-1, OH-2 and MM.1S as well as in primary
myeloma cells. Pharmacologic inhibition of the Janus
kinase/signal transducer and activator of transcription
(JAK/STAT) pathway abolished STAT3 phosphorylation
and SGK1 induction. In addition, small hairpin RNA
(shRNA)-mediated knock-down of STAT3 reduced basal
and induced SGK1 levels. Furthermore, downregulation
of SGK1 by shRNAs resulted in decreased proliferation
of myeloma cell lines and reduced cell numbers. On the
molecular level, this was reflected by the induction of
cell cycle inhibitory genes, for example, CDKNA1/p21,
whereas positively acting factors such as CDK6 and
RBL2/p130 were downregulated. Our results indicate that
Correspondence: Dr M Janz, Department of Hematology, Oncology
and Tumorimmunology, Charité, University Medical School Berlin,
Campus Virchow-Klinikum and Campus Buch, Max Delbrück Center
for Molecular Medicine, Robert-Rössle-Strae 10, Berlin 13092, Germany.
E-mail: [email protected]
Received 15 July 2010; revised 26 January 2011; accepted 10 February
2011; published online 11 April 2011
SGK1 is a highly cytokine-responsive gene in myeloma
cells promoting their malignant growth.
Oncogene (2011) 30, 3198–3206; doi:10.1038/onc.2011.79;
published online 11 April 2011
Keywords: multiple myeloma; cytokines; SGK1; STAT3
Introduction
Multiple myeloma (MM) is a malignancy of monoclonal
plasma cells that is typically localized to the bone
marrow. The tumor cells accumulate in this location not
only due to intrinsic properties of the malignant clone,
but also due to the fact that they depend on external
stimuli from the bone marrow microenvironment. In
addition to genetic aberrations, a multitude of bone
marrow-derived signals seem to be important for
sustaining the survival and growth of MM cells.
Although interleukin (IL)-6 is considered to be the most
potent growth factor for MM cells, a number of other
cytokines including IL-21, IL-15, tumor necrosis factor
(TNF), insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) have been reported to
stimulate their proliferation or to protect them against
apoptosis (Hideshima et al., 2007).
Several intracellular signaling pathways are known to
be activated in myeloma cells by external stimuli, notably
the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), the Ras/mitogen-activated
protein kinase (Ras/MAPK), the phosphoinositide-3
kinase (PI3K)/AKT and the nuclear factor kappa B pathway (Podar et al., 2009). However, less is known about
the transcriptional signatures of the various pathways.
We hypothesized that the intracellular signals evoked by
cytokines converge and regulate transcription of a set
SGK1 in multiple myeloma
U-M Fagerli et al
3199
of genes that are common targets for several growth
factors and therefore constitute pivotal mediators of the
tumor-promoting effects of autocrine or paracrine
stimuli. To identify such targets, we stimulated MM
cell lines with various cytokines and performed gene
expression profiling experiments. Among a limited
number of genes consistently activated in response to
all cytokines analyzed, SGK1, which encodes the serum
and glucocorticoid-regulated protein kinase 1 (SGK1),
was one of the most prominent target genes.
SGK1, which has been shown to be involved in
cellular proliferation and apoptosis protection, is a
serine/threonine kinase of the AGC (cAMP-dependent,
cGMP-dependent and protein kinase C) kinase family
that also includes AKT (Webster et al., 1993). The
SGK1 gene is under strict transcriptional control and
SGK1 mRNA expression is rapidly induced in response
to a variety of external stimuli (Webster et al., 1993;
Leong et al., 2003). In addition, SGK1 protein is
regulated at the posttranslational level by phosphorylation and subcellular localization. Similarly to AKT,
SGK1 is activated through phosphorylation by phosphoinositide-dependent protein kinase-1 (PDK-1), a
signaling intermediate downstream of PI3K (Kobayashi
and Cohen, 1999; Park et al., 1999). Several myeloma
growth factors, like IGF-1 and HGF, are known to
activate the PI3K/AKT pathway, and AKT kinase has
been shown to provide important growth and survival
signals for MM cells (Tu et al., 2000; Hideshima et al.,
2001; Hsu et al., 2001). AKT and SGK1 share several
substrates, but, as exemplified for the transcription
factor FKHRL1, they phosphorylate both common and
distinct residues in their substrates (Brunet et al., 2001).
It is therefore possible that AKT and SGK1 have complementary rather than redundant roles in regulating cell
growth and survival. The diverse and complex regulation of SGK1 expression and activation by external
signals indicates that this kinase is a pivotal mediator of
the cellular response to environmental stimuli.
In this study, we show that in myeloma cells SGK1 is
rapidly and strongly induced by growth and survival
factors expressed in the bone marrow, that this induction is mediated by the JAK/STAT signaling pathway,
and that downregulation of SGK1 by RNA interference
results in impaired proliferation of myeloma cell lines.
Results and discussion
To gain insight into the transcriptional changes in
MM cells induced by growth and survival factors of
the bone marrow microenvironment, we determined the
effect of cytokine stimulation on the gene expression
profile of myeloma cell lines. The cell lines IH-1, OH-2
and INA-6 were cultured in the absence or presence
of IL-6 (all cell lines), TNFa (OH-2 cells), IL-21 (OH-2
cells) or IL-6 in combination with HGF (IH-1 cells).
In addition, INA-6 cells were co-cultured with bone
marrow stromal cells with or without the IL-6 receptor
antagonist Sant7 to assess the impact of IL-6 signaling
in the context of stromal cells. Oligonucleotide microarray analyses were performed to identify genes that
were significantly upregulated in cytokine-stimulated
myeloma cells compared with unstimulated cells.
A limited set of genes was consistently induced in
IH-1 and OH-2 cells by all cytokines analyzed, among
them JUNB, BCL6, RUNX3, BCL3, ETV6, ICAM1,
MIR21 as well as the gene encoding SGK1 (Supplementary Table 1a). SGK1 expression was also highly
dependent on IL-6 in INA-6 cells, indicating that SGK1
is a prominent transcriptional target of cytokineinduced signaling in myeloma cells (Supplementary
Tables 1b and c; for an overview of SGK1 expression
in IH-1, OH-2 and INA-6 cells under different culture
conditions see Supplementary Table 1d). As SGK1
exhibits mitogenic and antiapoptotic properties in other
cellular systems (Brunet et al., 2001; Leong et al., 2003)
and is a component of the PI3K pathway (Kobayashi
and Cohen, 1999; Park et al., 1999), which mediates
important survival signals for myeloma cells, we decided
to analyze the role of SGK1 in MM in more detail.
To confirm and extend the results of our microarray
experiments, we analyzed SGK1 mRNA expression and
the kinetics of SGK1 mRNA induction in various MM
cell lines following cytokine stimulation (IH-1, OH-2,
INA-6, ANBL-6 and MM.1S cells). We could demonstrate that in IL-6-dependent and IL-6-independent cell
lines SGK1 mRNA was strongly and rapidly induced by
a number of MM growth-promoting and anti-apoptotic
cytokines, including IL-6, IL-15, IL-21 and TNFa, as
well as by co-culture with bone marrow stromal cells
(Figures 1a–c and Supplementary Figure 1). These
results suggested that the immediate and robust induction of SGK1 by cytokines, in particular by IL-6, is a
recurrent feature of cytokine-responsive myeloma cell
lines. To permit a comparison of SGK1 mRNA
expression between myeloma cell lines and other
lymphoid-derived cell lines, we carried out real-time
PCR on a panel of cell lines that were derived from Band T-cell malignancies of distinct differentiation stages.
This analysis demonstrated a more prominent expression of SGK1 in MM cell lines compared with a variety
of B- and T-cell leukemia/lymphoma cell lines, further
indicating that SGK1 may be of particular function for
myeloma cells (Supplementary Figure 2).
We next verified in IH-1 and OH-2 cells the induction
of SGK1 by cytokines at the protein level and compared
this with the induction of proliferation, respectively.
Cells were treated with IL-6, IL-21, IL-15, TNFa, HGF
or IGF-1 before harvesting for SGK1 western blot
analysis and measurement of DNA synthesis reflecting
cell division. In general, induction of SGK1 expression
closely correlated, albeit not absolutely, with the mitogenic effect of different cytokines on growth factordependent myeloma cells (Figure 1d). This strongly
suggested a central role of SGK1 in the proliferative
response to external stimuli.
To verify SGK1 mRNA expression in primary
myeloma cells and to investigate whether the strong
regulation of SGK1 by IL-6 observed in cell lines could
be corroborated in primary tumor material, we isolated
Oncogene
SGK1 in multiple myeloma
U-M Fagerli et al
5
−
−
+
+ IL-15
+ IL-21
IGF-1
TNF
HGF
IL-15
IL-21
untreated
IGF-1
HGF
SGK1
+ IL-6
control
−
90'
Sant7
60'
+
30'
−
+
90'
−
−
60'
−
−
1
30'
+
BMSCs
untreated
IL-6
TNF
IL-15
IL-6
IL-21
1
2
90'
2
3
IL-6
3
4
60'
relative SGK1 mRNA expression
4
OH-2
30'
IH-1
5
untreated
relative SGK1 mRNA expression
3200
SGK1
NB
RT-PCR
GAPDH
GAPDH
1
2
3
4
INA-6
MM.1S
IH-1
OH-2
SGK1
SGK1
WB
WB
p42/44
GAPDH
control IL-6 IL-21 IL-15 TNF HGF IGF-1
3000
[3H]-thymidine
incorporation
[3H]-thymidine
incorporation
control IL-6 IL-21 IL-15 TNF HGF IGF-1
17500
15000
12500
10000
7500
5000
2500
control IL-6 IL-21 IL-15 TNF HGF IGF-1
2000
1000
control IL-6 IL-21 IL-15 TNF HGF IGF-1
Figure 1 SGK1 expression is induced by cytokines in myeloma cell lines and parallels the cytokine-induced proliferation of myeloma
cells. (a) Quantitative PCR analysis of SGK1 mRNA expression in IH-1 and OH-2 cells following stimulation with IL-6 (5 ng/ml),
IL-21 (20 ng/ml), IL-15 (20 ng/ml), TNFa (10 ng/ml), HGF (150 ng/ml) or IGF-1 (100 ng/ml) for 24 h. SGK1 transcript levels were
normalized to b-actin expression in each sample and are presented as fold induction relative to SGK1 expression in unstimulated cells,
set as 1. Error bars indicate s.d. of at least three independent experiments. Details of cell lines, cell culture conditions and SGK1 primer
sequences are available in Supplementary Materials and Methods. (b) Analysis of SGK1 mRNA expression by northern blotting (NB)
in INA-6 cells. RNAs were isolated from INA-6 cells cultured with IL-6 (lane 1) or without IL-6 for 12 h (lane 2) and INA-6 cells
co-cultured with bone marrow stromal cells (BMSCs) in the absence (lane 3) or presence (lane 4) of the IL-6 receptor antagonist
Sant7 for 12 h (see Supplementary Materials and Methods for isolation of BMSCs and co-culture conditions). In all, 10 mg of total
RNA, extracted with the RNeasy kit (Qiagen, Hilden, Germany), were subjected to denaturing gel electrophoresis and transferred to a
nylon membrane (Appligene, Heidelberg, Germany). DNA probes for detection of SGK1 or glyceraldehyde 3-phosphate dehydrogenase
(GAPDH; control) mRNA were labeled with [a-32P]dCTP by random priming (Fermentas, St Leon-Rot, Germany). Membrane
hybridization and washing was carried out under high-stringency conditions (ExpressHyb solution; Clontech, Heidelberg, Germany).
(c) SGK1 transcript levels in MM.1S cells were determined by RT–PCR following stimulation with IL-6 (2 ng/ml), IL-15 (50 ng/ml) or
IL-21 (100 ng/ml) for the indicated time periods. See Supplementary Materials and Methods for primer sequences. (d) Upper panel:
SGK1 protein expression was determined by western blot (WB) analysis (Cell Signaling Technology, Frankfurt am Main, Germany;
#3272) in IH-1 and OH-2 cells after stimulation with IL-6 (5 ng/ml), IL-21 (20 ng/ml), IL-15 (20 ng/ml), TNFa (10 ng/ml), HGF
(150 ng/ml) or IGF-1 (100 ng/ml) for 18 h. Expression of p42/44 (Cell Signaling Technology, #9102) and GAPDH (Abcam, Cambridge,
UK, ab9484) protein was analyzed to control for equal loading. Lower panel: DNA synthesis in cytokine-stimulated IH-1 and OH-2
cells was determined by [3H]-thymidine incorporation assays. Cells were cultured in 96-well plates with 3 104 cells per well, treated
with the indicated cytokines for 48 h and pulsed for additional 18 h with 0.037 Mbq [3H]-thymidine before harvesting. Measurements
were performed in triplicate and are expressed as means±s.d. Data are from one of at least four independent experiments.
myeloma cells from bone marrow aspirates of patients
with MM. Primary cells were cultured in the presence or
absence of IL-6 signaling. In the latter case, this was
achieved either by withdrawal of IL-6 from the culture
and/or by addition of the IL-6 receptor antagonist Sant7
to ensure complete blockade of IL-6-mediated effects
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induced either by the exogenously added or any endogenously produced IL-6. In the majority of cases (12/14
patients), culture conditions without IL-6 or blocking of
IL-6 signaling by Sant7 significantly reduced SGK1 transcript levels, demonstrating that SGK1 expression in primary
tumor cells is highly responsive to IL-6 (Figures 2a and b).
SGK1 in multiple myeloma
U-M Fagerli et al
1.0
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
0.2
0.0
***
0.8
0.6
0.2
0.0
0.0
0.0
MM #2
MM #4
n.s.
1.2
1.2
1.2
1.0
1.0
1.0
1.0
0.8
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
MM #8
1.0
0.8
0.8
0.8
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0.2
0.2
0.0
0.0
0.0
0.0
MM #10
MM #11
L-
6
-6
+
IL
+ IL-6
S7
6
L-
***
0.4
+
-I
-6
***
IL
IL
+
6
L-
+ IL-6
S7
+
-I
-6
IL
+
MM #9
n.s.
0.6
***
+
***
0.6
+
0.8
+ IL-6
S7
1.2
1.0
1.0
-6
1.2
1.0
1.2
+ IL-6
S7
-6
IL
+
6
L-
MM #7
n.s.
1.2
+ IL-6
S7
+
MM #6
n.s.
0.2
0.0
IL
+
+ IL-6
S7
L-I
+
+
IL
+ IL-6
S7
IL
+
MM #5
***
0.4
***
-6
0.0
6
0.2
0.0
-6
0.2
0.0
-6
0.2
1.2
+
0.4
**
-I
**
0.6
**
***
IL
-6
-I
L6
+
I
L
+ -6
S7
MM #3
-I
MM #1
***
+
+
+
6
IL
-
IL
-6
-I
L6
+
I
L
+ -6
S7
0.2
+ IL-6
S7
0.4
0.2
+
6
IL
-
1.0
***
0.4
+
+
***
1.2
+ IL-6
S7
1.2
1.0
n.s.
+
1.2
1.0
+
1.2
+ IL-6
S7
SGK1 mRNA expression
relative to GAPDH
3201
MM #12
p = 0.0075
1.2
1.0
1.0
0.2
0.0
0.0
-6
IL
+
L-I
IL
+
MM #13
L-
0.4
0.2
6
0.4
-6
0.6
6
+
+ IL-6
S7
0.8
0.6
*
8
6
4
-I
***
0.8
10
*
CT SGK1 - GAPDH
1.2
MM #14
+ IL-6
+ IL-6 + Sant7
Figure 2 IL-6 is a potent stimulus for SGK1 mRNA expression in primary myeloma cells. (a) Quantitative PCR analysis of SGK1
mRNA expression in malignant plasma cells isolated from the bone marrow of MM patients. CD138-positive MM cells were purified
from routine diagnostic bone marrow aspirates by immunomagnetic bead separation using MACS MicroBeads (Miltenyi Biotec,
Bergisch Gladbach, Germany). Primary tumor cells derived from 14 different donors were cultured after purification in RPMI 1640
medium supplemented with 20% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, 2 mM
glutamine for 18–24 h with or without IL-6 stimulation (IL-6, 2 ng/ml). In addition, where indicated, IL-6 signaling was blocked by
treatment with the IL-6 receptor antagonist Sant7 (S7; 50 mg/ml). SGK1 and GAPDH mRNA levels were assessed by real-time PCR
and relative SGK1 expression was calculated using the 2DDCt method with SGK1 expression in IL-6-treated cultures set as 1. Error
bars denote 95% confidence intervals. n.s., not significant; *Po0.05; **Po0.01; ***Po0.001. The use of primary human cells was
approved by the local ethics committees of the participating institutions. (b) Box plot analysis comparing the DCt values for SGK1 and
GAPDH mRNA from all samples treated with IL-6 versus all samples treated with IL-6 plus the IL-6 antagonist Sant7. The boxes
represent the 25th, 50th (median) and 75th percentile of the two groups, respectively. Notches indicate an estimate of the 95%
confidence interval of the respective median; the whiskers delineate the minimum and maximum of all data. Note that a smaller DCt
value (as can be seen for the group of IL-6-treated samples) represents a smaller difference between SGK1 and GAPDH Ct values,
reflecting a stronger SGK1 expression across all the samples and vice versa. The two groups (IL-6 vs IL-6 þ Sant7) show a statistically
significant difference with a P-value of 0.0075 as determined by a one-sided Welch’s t-test.
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SGK1 in multiple myeloma
U-M Fagerli et al
3202
To investigate which pathways mediate the induction
of SGK1, we performed IL-6 stimulation experiments in
conjunction with the use of small molecule inhibitors or
specific downregulation of signaling molecules by RNA
interference. INA-6 cells cultured in the presence of
IL-6 displayed high levels of STAT3 phosphorylation,
whereas only small amounts of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2; p42/44)
were detectable (Figure 3a). IL-6 withdrawal resulted in
a complete loss of phospho-STAT3 without any effects
on the ERK1/2 phosphorylation status. Re-stimulation
with IL-6 led to rapid phosphorylation of STAT3 and
ERK1/2, which could be blocked efficiently with the
JAK inhibitor P6 and the MEK1/2 inhibitor U0126,
respectively. Whereas the inhibition of MAP kinase
activation had no effect on SGK1 mRNA, the blockade
of JAK/STAT signaling and concomitant STAT3
phosphorylation abolished SGK1 mRNA upregulation
(Figure 3a). Essentially identical results were observed
with the cell lines MM.1S, IH-1 and OH-2 (Supplementary Figure 3). To confirm these observations by an
independent approach, we used a small hairpin RNA
(shRNA) expression construct that inhibits STAT3
expression. As STAT3 knock-down induces apoptosis
in INA-6 cells, we chose a time frame in which STAT3
expression is efficiently downregulated without already
compromising overall cell survival. Transfection of
INA-6 cells with the STAT3-directed shRNA plasmid
resulted in a substantial downregulation of basal and
IL-6-induced SGK1 mRNA levels (Figure 3b). Similarly,
knock-down of STAT3 in MM.1S cells, which do not
depend on STAT3 for survival, inhibited SGK1 mRNA
induction by IL-6 (Figure 3c), and SGK1 mRNA
induction could be rescued by ectopic expression of a
mutated STAT3 protein that is resistant to shRNAmediated downregulation (Figure 3d). Taken together,
these data indicate that transcriptional activation of
the SGK1 gene is mediated by JAK/STAT signaling in
myeloma cells.
At this point, our results suggested a role for SGK1 in
promoting growth and survival of myeloma cells. To
test this, we selectively blocked SGK1 expression by
RNA interference. Employing a vector-based shRNA
expression system, we identified several constructs that
downregulated SGK1 mRNA expression to varying
extents (Supplementary Figure 4). Next, we analyzed
whether shRNA-mediated knock-down of SGK1 in
myeloma cells affects their proliferation and viability.
The transient transfection experiments were performed
with INA-6 and AMO-1 myeloma cells. AMO-1 cells,
which demonstrate constitutive SGK1 expression
(Figure 4a; Supplementary Figure 2), were included in
these and the following experiments since they can be
efficiently electroporated and purified for functional
assays. Downregulation of SGK1 resulted in a marked
reduction of DNA synthesis in INA-6 cells and a
moderate decrease in AMO1 cells compared with
control-transfected cells (Figure 4a). To assess the
effects of a prolonged SGK1 knock-down in myeloma
cells, we used shRNA vector constructs that are
propagated by extrachromosomal replication and carry
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a puromycin resistance gene, thus allowing for selection
of shRNA-expressing cells. Transfection of SGK1
shRNA constructs and subsequent antibiotic selection
led to a significant decrease in cell numbers of viable
AMO-1 myeloma cells, as determined by annexin Vfluorescein isothiocyanate/propidium iodide staining
and flow cytometry (Figure 4b). To gain insight into
the molecular mechanisms that are influenced by SGK1,
we performed gene expression profiling experiments of
INA-6 cells following shRNA-mediated knock-down of
SGK1. Gene set enrichment analysis demonstrated that
downregulation of SGK1 is significantly associated with
changes in the expression levels of genes that are involved
in cell cycle regulation (Supplementary Figure 5). From
this gene set, we selected genes that showed a high rank
metric score and are known to be important regulators
of cell cycle progression, such as CDK6, RBL2/p130,
CDKN1A/p21, CDKN1B/p27 and CDKN2D/p19, and
examined their expression in control and SGK1
shRNA-treated samples. By quantitative PCR, we were
able to verify their negative or positive regulation
after SGK1 knock-down (Figure 4c). Together with
our observation that the proliferative response of
the myeloma cell lines IH-1 and OH-2 closely correlates with SGK1 expression (Figure 1d), these data
indicate a role of SGK1 in cell cycle regulation of
myeloma cells.
Numerous growth factors and cell–cell interactions
have been described to promote proliferation and
survival of myeloma cells in the bone marrow microenvironment. The biological impact for disease initiation, progression and resistance to therapy is likely to be
determined by the sum of these complex interactions
(Hideshima et al., 2007; Podar et al., 2009), implying
that the inhibition of single factors or signaling
molecules could be compensated for by other, redundant stimuli. As a consequence, treatment strategies
targeted against single cytokines or even against specific
intracellular signaling intermediates may be of limited
clinical activity, as already indicated by early attempts
to block IL-6-mediated signaling in MM (van Zaanen
et al., 1998; Ocio et al., 2008). We hypothesized that
growth-promoting cytokines redundantly induce the
expression of proteins that exert a central function
for the malignant growth of myeloma cells. If this
assumption is correct, directing treatment against such
molecules might be more effective than attacking
signaling events that lead to the expression of these
factors.
Here, we argue that SGK1 is a promising candidate
for such a critical factor in myeloma cells. Our study
shows that induction of the SGK1 gene is a prominent
feature of the transcriptional response to cytokines,
that upregulation of SGK1 is correlated with enhanced
proliferation, and that shRNA-mediated downregulation of SGK1 results in a significant reduction in DNA
synthesis and in the number of viable cells. On the
molecular level, SGK1 silencing is accompanied by
the induction of cell cycle inhibitory genes as well as
the downregulation of positive regulators of cell cycle
progression.
SGK1 in multiple myeloma
U-M Fagerli et al
p-p42/44
p-STAT3
WB
WB
STAT3
p42/44
SGK1
SGK1
RT-PCR
RT-PCR
GAPDH
GAPDH
GAPDH
U
pS STA
U
-S T3TA 2
T3
-3
U
pS
WB
STAT3
RT-PCR
-actin
GAPDH
INA-6
MM.1S + IL-6
MM.1S + IL-6
U
pS
pS
U
+
pc
D
pS
U
+
pS pc
D
U
N
+
pS pS A3
3
U
-S RpS TA HA
T
U
-S 3-2
TA
+
T3 pc
D
-2
N
+
A
pS 3
3R
-H
A
INA-6
N
pS + p A3
U S3
-S R
pS T -H
U AT A
co -ST 3-2
nt A
ro T3 + p
l -2 cD
N
+
pS A3
3R
-H
A
pS
pSU-STAT3-3
control
pSU-STAT3-2
-S
U
WB
-tubulin
p-STAT3
SGK1
STAT3
RT-PCR
pSU
-2
T3
SGK1
pS
pS
U
TA
control
- IL-6
+ IL-6 30'
+ IL-6 30'
pSUSTAT3-2
+ IL-6
- IL-6
+ IL-6
INA-6
not transfected
INA-6
pSU
+ IL-6 120'
+ IL-6 30'
+ IL-6 60'
U0126
+ IL-6 120'
+ IL-6 60'
+ IL-6 30'
- IL-6 o/n
+ IL-6
+ P6 120'
+ IL-6 120'
DMSO
+ IL-6 60'
+ IL-6 30'
+ IL-6 120'
+ IL-6 60'
+ IL-6 30'
- IL-6 o/n
+ IL-6
P6
+ U0126 120'
3203
DMSO
SGK1
p-STAT3
STAT3
RT-PCR
WB
HA
GAPDH
MM.1S + IL-6
-actin
MM.1S + IL-6
Figure 3 Inhibition of the JAK/STAT pathway blocks SGK1 induction by IL-6. (a) Left: blocking of the JAK/STAT signal
transduction pathway by the JAK inhibitor P6. INA-6 cells, deprived of IL-6 overnight, were treated with 1 mM P6 (Calbiochem,
Darmstadt, Germany, #420099) or the corresponding amount of solvent (dimethylsulphoxide, DMSO) 30 min before IL-6 stimulation.
Cells were harvested 30, 60 and 120 min after addition of IL-6 and analyzed for STAT3 total protein expression (Cell Signaling
Technology, #9132) and STAT3 phosphorylation (p-STAT3; Cell Signaling Technology, #9131) by western blotting (WB). SGK1 and
GAPDH (control) mRNA expression was determined by reverse transcriptase (RT)–PCR. Right: blocking of the mitogen-activated
protein (MAP) kinase pathway by the MEK1/2 inhibitor U0126. IL-6-starved INA-6 cells were incubated with 20 mM U0126
(Calbiochem, #662005) or DMSO for 30 min, stimulated with IL-6 and harvested at the indicated time points. WB: phosphorylated
(p-p42/44; Cell Signaling Technology, #9101) and total p42/44 (Cell Signaling Technology, #9102) protein expression, RT–PCR: SGK1
and GAPDH mRNA expression. Protein and mRNA data were derived in parallel from the same experiment. (b) INA-6 cells were
electroporated (GenePulser II, Bio-Rad, Munich, Germany; 960 mF, 0.27 kV) with control vector (pSU, 40 mg/ml and 5 106 cells per
electroporation cuvette) or STAT3-directed shRNA vector (pSU-STAT3-2, 40 mg/ml) and an enhanced green fluorescent protein
(EGFP) expression plasmid (pEGFP-N3, Clontech; 20 mg/ml). EGFP-positive cells were purified by fluorescence-activated cell sorting
(FACS) after 44 h and either cultured further on with IL-6 ( þ IL-6), deprived of IL-6 for 6 h (IL-6) or deprived of and re-stimulated
with IL-6 for 30 min ( þ IL-6 300 ). SGK1 and GAPDH transcripts were analyzed by RT–PCR (left). STAT3 protein knock-down was
verified by western blot analysis (right; anti a-tubulin, Santa Cruz Biotechnology, Heidelberg, Germany; H-300, sc-5546). For shRNA
vector construction and RNA interference target sequences see Supplementary Materials and Methods. (c) MM.1S cells were
electroporated (GenePulser II, Bio-Rad, 950 mF, 0.3 kV) with STAT3 shRNA constructs (pSU-STAT3-2 or pSU-STAT3-3, 40 mg/ml)
or control vector (pSU, 40 mg/ml) in combination with an EGFP expression plasmid (20 mg/ml) and purified by FACS after 24 h.
At 72 h post transfection, cells were stimulated with IL-6 (2 ng/ml) for 60 min. SGK1 and GAPDH mRNA expression was determined
by RT–PCR. Western blot analysis was performed to control for efficient STAT3 protein knock-down. b-Actin, loading control
(Sigma, Taufkirchen, Germany, A5316). (d) MM.1S cells were transfected with the indicated combinations of control vectors (pSU,
pcDNA3), STAT3-directed shRNA vector (pSU-STAT3-2) and/or an expression plasmid encoding a small interfering RNA (siRNA)resistant, HA-tagged STAT3 protein (pS3R-HA, 10 mg/ml) along with an EGFP expression plasmid. FACS-purified cells were
stimulated after 72 h with IL-6 for 60 min. RT–PCR: SGK1 and GAPDH mRNA expression. WB: expression of endogenous and
ectopically expressed STAT3 protein (anti-HA, Covance, Princeton, NJ, USA, clone 16B12, #MMS-101P). The expression plasmid
for siRNA-resistant, HA-tagged STAT3 has been described previously (Chatterjee et al., 2004).
Oncogene
SGK1 in multiple myeloma
U-M Fagerli et al
3204
n.s.
40 000
*
30 000
*
*
*
20 000
SGK1
354-372
SGK1
896-914
SGK1
247-265
SGK1
192-210
6 000
4 000
2 000
shRNA
E2 se
F rt
SG -4
K
SG 1 1
K 92
SG 1 2 -21
K 47 0
SG 1 8 -2
6
K 96 5
1 -9
35 1
4- 4
37
2
no
*
8 000
in
no insert
shRNA
E2F-4
10 000
*
10 000
354-372
50 000
12 000
scrambled
no
sc ins
r e
E2 am rt
E2F-4
F bl
SG -4 ed
SGK1
SGK1
K 896
1 -9
896-914
35 1
4- 4
37
2
SGK1
INA-6
AMO-1
no insert
n.s.
60 000
[3H]-thymidine incorporation
3
[ H]-thymidine incorporation
14 000
SGK1
SGK1
GAPDH
GAPDH
RT-PCR
RT-PCR
13
n.s.
90
80
11
AMO-1
3
60
50
40
*
30
20
*
2.5
2
10
shRNA control (Luc)
shRNA SGK1 354-372
**
*
1
0.5
9
***
**
shRNA SGK1 896-914
1.5
SGK1
896-914
E2F-4
SGK1
354-372
Luc
no insert
no DNA
10
mRNA expression
relative to GAPDH
viable cells (%)
70
shRNA
***
12
*** ***
SGK1
***
**
CDK6
n.s.
***
3
2
**
RBL2
(p130)
4
1
CDKN2D
(p19)
CDKN1B
(p27)
CDKN1A
(p21)
Figure 4 Knock-down of SGK1 by RNA interference reduces proliferation and viability of myeloma cells. (a) Effect of SGK1 knockdown on proliferation of INA-6 (left panel) and AMO-1 (right panel) cells. Cells were electroporated (GenePulser II, Bio-Rad, 960 mF
and 0.27 kV for INA-6 cells, 950 mF and 0.25 kV for AMO-1 cells) with the indicated pSUPER shRNA plasmids (40 mg/ml) along with
an enhanced green fluorescent protein (EGFP) expression plasmid (10 mg/ml), purified by fluorescence-activated cell sorting (FACS)
after 48 h and pulsed with [3H]-thymidine (0.037 Mbq) for 24 h. Data are presented as c.p.m. of [3H]-thymidine incorporation (triplicate
measurements, means±s.d.). n.s., not significant; *statistical significance at Po0.001 (INA-6) and Po0.01 (AMO-1) to control vectors
(no insert, scrambled, E2F-4). In parallel, SGK1 and GAPDH transcripts were analyzed by RT–PCR in the respective cell populations.
Data are representative of four (INA-6) or three (AMO-1) experiments. See Supplementary Materials and Methods for shRNA target
sequences. (b) Effect of sustained SGK1 downregulation on myeloma cell viability. AMO-1 cells were transfected with control and
SGK1-directed pRepH1 shRNA vectors (40 mg/ml) and selected for shRNA-expressing cells by the addition of puromycin (1.5 mg/ml)
24 h after electroporation. The percentage of viable cells was determined by exclusion of dead and apoptotic cells by flow cytometry
following staining with annexin V- fluorescein isothiocyanate (FITC) (Bender MedSystems, Vienna, Austria) and propidium iodide
(PI). The fraction of viable cells, negative for annexin V and PI, is expressed as percentage of total cells in the flow cytometry analysis.
Measurements were performed in triplicate. Error bars denote s.d.; n.s., not significant; *statistical significance at Po0.001 to control
vectors (no insert, Luc, E2F-4). Data are representative of five independent experiments. (c) Impact of SGK1 knock-down on cell cycle
regulatory genes. INA-6 cells were electroporated with the indicated shRNAs and an EGFP expression vector, purified after 48 h
by FACS and harvested for RNA isolation. Expression of SGK1, CDK6, RBL2, CDKN1A, CDKN1B and CDKN2D mRNA was
analyzed by quantitative PCR and relative expression levels were calculated using the 2DDCt method with the control shRNA sample
defined as 1. Error bars show 95% confidence intervals. n.s. denotes not significant; *Po0.05; **Po0.01; ***Po0.001. Primer sequences
are listed in Supplementary Materials and Methods.
Our finding that SGK1 is a cytokine-responsive and
growth-supporting gene in malignant plasma cells, is in
line with previous reports that described a strong SGK1
expression or induction by growth factors in other
tumor entities. SGK1 is upregulated in breast and in
prostate cancer cells, promoting apoptosis resistance
Oncogene
(Wu et al., 2004; Shanmugam et al., 2007). In breast
cancer tissue, SGK1 expression demonstrated a significant correlation with the presence of activated,
phosphorylated AKT protein, suggesting a common
involvement of both kinases in the PI3K pathway, that
is, PI3K might activate SGK1 and AKT in parallel
SGK1 in multiple myeloma
U-M Fagerli et al
3205
(Sahoo et al., 2005). PI3K-mediated signaling events
have an important oncogenic role in myeloma cells.
The PI3K/AKT pathway is activated by a number of
bone marrow-derived growth factors, most notably
IL-6 and IGF-1, and inhibition of PI3K or AKT
induces cell cycle arrest and apoptosis of myeloma
cells (Tu et al., 2000; Hideshima et al., 2001; Hsu
et al., 2001). However, we recently observed that only
a proportion of MM cases are sensitive to AKT
inhibition (Zöllinger et al., 2008). It is currently unclear
whether AKT-independent myeloma cells are completely independent of PI3K-derived signals or whether
other downstream signaling components, among them
potentially SGK1, can substitute for AKT activity
in these tumors. In this context, it is of particular
interest that, using a broad panel of carcinoma cell
lines, it has been demonstrated that in the absence of
AKT activation PI3K transmits alternative signals
to downstream substrates such as the SGK family
member SGK3 (Vasudevan et al., 2009). Given these
observations, it will be important to determine the
exact contribution of both kinases, AKT and SGK1, to
the malignant growth of myeloma cells.
In contrast to most protein kinases, which are
constitutively expressed, transcription of the SGK1 gene
is subject to regulation by extracellular signals (Webster
et al., 1993; Leong et al., 2003). In myeloma cells, SGK1
was upregulated by a number of growth factors as well
as bone marrow stromal cells, with IL-6 representing the
most potent stimulus. IL-6 can activate the JAK/STAT,
MAPK and PI3K/AKT pathway in myeloma cells
(Ogata et al., 1997; Catlett-Falcone et al., 1999; Tu
et al., 2000). Our experiments indicate that IL-6 induces
SGK1 transcription primarily through the JAK/STAT
cascade, which is supported by a study in which SGK1
was listed in the group of STAT3-dependent target
genes in MM cells (Brocke-Heidrich et al., 2004).
In cholangiocarcinoma cells, IL-6 induces p38 MAPK
activation that in turn not only stimulates SGK1 phosphorylation and nuclear translocation, but also SGK1
expression (Meng et al., 2005). On the basis of the
inhibitor experiments, however, we found no evidence
for an involvement of p38 in SGK1 induction in
myeloma cells.
Taken together, our findings provide evidence for a
scenario in which SGK1 represents a functional
convergence point between the transcriptional response
to external signals and intracellular phosphorylation
cascades. Induction of the SGK1 gene by growth factors
could in turn amplify the cellular response to extracellular stimuli by subsequent participation of the SGK1
protein kinase in growth-associated signaling events.
Thus, SGK1 represents an attractive candidate for
further evaluation as a therapeutic target in MM.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Brigitte Wollert-Wulf, Mandy Terne, Pia Herrmann
(Berlin), Berit Strdal and Hanne Hella (Trondheim) for
excellent technical assistance and Hans-Peter Rahn (Berlin) for
cell sorting. Reuven Agami (Amsterdam, The Netherlands)
kindly provided the pSUPER vector and Matthias Truss
(Berlin) the pRepH1 construct. This work was supported by
grants from the Deutsche Krebshilfe (10-2225-Ja 1), the Berlin
Cancer Society, the Deutsche Forschungsgemeinschaft (KFO
216 to TS, MC and RCB), the Norwegian Cancer Society,
the Cancer Fund of St Olavs Hospital, Trondheim, and the
Research Council of Norway.
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Oncogene