Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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-α, 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 cytokine-induced 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 SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their malignant growth.

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 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), TNFα (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 cytokine-induced 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 TNFα, 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 B- and 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).

Figure 1
figure1

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), TNFα (10 ng/ml), HGF (150 ng/ml) or IGF-1 (100 ng/ml) for 24 h. SGK1 transcript levels were normalized to β-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 μg 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 [α-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), TNFα (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.

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, TNFα, 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 factor-dependent 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 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 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).

Figure 2
figure2

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 μg/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 μg/ml). SGK1 and GAPDH mRNA levels were assessed by real-time PCR and relative SGK1 expression was calculated using the 2−ΔΔCt method with SGK1 expression in IL-6-treated cultures set as 1. Error bars denote 95% confidence intervals. n.s., not significant; *P<0.05; **P<0.01; ***P<0.001. The use of primary human cells was approved by the local ethics committees of the participating institutions. (b) Box plot analysis comparing the ΔCt 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 ΔCt 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 + Sant7) show a statistically significant difference with a P-value of 0.0075 as determined by a one-sided Welch's t-test.

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 shRNA-mediated downregulation (Figure 3d). Taken together, these data indicate that transcriptional activation of the SGK1 gene is mediated by JAK/STAT signaling in myeloma cells.

Figure 3
figure3

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 μM 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 μM 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 μF, 0.27 kV) with control vector (pSU, 40 μg/ml and 5 × 106 cells per electroporation cuvette) or STAT3-directed shRNA vector (pSU-STAT3-2, 40 μg/ml) and an enhanced green fluorescent protein (EGFP) expression plasmid (pEGFP-N3, Clontech; 20 μg/ml). EGFP-positive cells were purified by fluorescence-activated cell sorting (FACS) after 44 h and either cultured further on with IL-6 (+IL-6), deprived of IL-6 for 6 h (−IL-6) or deprived of and re-stimulated with IL-6 for 30 min (+IL-6 30′). SGK1 and GAPDH transcripts were analyzed by RT–PCR (left). STAT3 protein knock-down was verified by western blot analysis (right; anti α-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 μF, 0.3 kV) with STAT3 shRNA constructs (pSU-STAT3-2 or pSU-STAT3-3, 40 μg/ml) or control vector (pSU, 40 μg/ml) in combination with an EGFP expression plasmid (20 μg/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. β-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 μg/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).

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 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 V-fluorescein 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.

Figure 4
figure4

Knock-down of SGK1 by RNA interference reduces proliferation and viability of myeloma cells. (a) Effect of SGK1 knock-down on proliferation of INA-6 (left panel) and AMO-1 (right panel) cells. Cells were electroporated (GenePulser II, Bio-Rad, 960 μF and 0.27 kV for INA-6 cells, 950 μF and 0.25 kV for AMO-1 cells) with the indicated pSUPER shRNA plasmids (40 μg/ml) along with an enhanced green fluorescent protein (EGFP) expression plasmid (10 μg/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 P<0.001 (INA-6) and P<0.01 (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 μg/ml) and selected for shRNA-expressing cells by the addition of puromycin (1.5 μg/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 P<0.001 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 2−ΔΔCt method with the control shRNA sample defined as 1. Error bars show 95% confidence intervals. n.s. denotes not significant; *P<0.05; **P<0.01; ***P<0.001. Primer sequences are listed in Supplementary Materials and Methods.

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.

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 (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 (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.

References

  1. Brocke-Heidrich K, Kretzschmar AK, Pfeifer G, Henze C, Löffler D, Koczan D et al. (2004). Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation. Blood 103: 242–251.

    CAS  Article  Google Scholar 

  2. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME . (2001). Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21: 952–965.

    CAS  Article  Google Scholar 

  3. Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, Savino R et al. (1999). Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 10: 105–115.

    CAS  Article  Google Scholar 

  4. Chatterjee M, Stühmer T, Herrmann P, Bommert K, Dörken B, Bargou RC . (2004). Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells. Blood 104: 3712–3721.

    CAS  Article  Google Scholar 

  5. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC . (2007). Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer 7: 585–598.

    CAS  Article  Google Scholar 

  6. Hideshima T, Nakamura N, Chauhan D, Anderson KC . (2001). Biologic sequelae of interleukin-6 induced PI3-K/Akt signaling in multiple myeloma. Oncogene 20: 5991–6000.

    CAS  Article  Google Scholar 

  7. Hsu J, Shi Y, Krajewski S, Renner S, Fisher M, Reed JC et al. (2001). The AKT kinase is activated in multiple myeloma tumor cells. Blood 98: 2853–2855.

    CAS  Article  Google Scholar 

  8. Kobayashi T, Cohen P . (1999). Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J 339 (Part 2): 319–328.

    CAS  Article  Google Scholar 

  9. Leong ML, Maiyar AC, Kim B, O'Keeffe BA, Firestone GL . (2003). Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells. J Biol Chem 278: 5871–5882.

    CAS  Article  Google Scholar 

  10. Meng F, Yamagiwa Y, Taffetani S, Han J, Patel T . (2005). IL-6 activates serum and glucocorticoid kinase via p38alpha mitogen-activated protein kinase pathway. Am J Physiol Cell Physiol 289: C971–C981.

    CAS  Article  Google Scholar 

  11. Ocio EM, Mateos MV, Maiso P, Pandiella A, San-Miguel JF . (2008). New drugs in multiple myeloma: mechanisms of action and phase I/II clinical findings. Lancet Oncol 9: 1157–1165.

    CAS  Article  Google Scholar 

  12. Ogata A, Chauhan D, Urashima M, Teoh G, Treon SP, Anderson KC . (1997). Blockade of mitogen-activated protein kinase cascade signaling in interleukin 6-independent multiple myeloma cells. Clin Cancer Res 3: 1017–1022.

    CAS  PubMed  Google Scholar 

  13. Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, Hemmings BA . (1999). Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18: 3024–3033.

    CAS  Article  Google Scholar 

  14. Podar K, Chauhan D, Anderson KC . (2009). Bone marrow microenvironment and the identification of new targets for myeloma therapy. Leukemia 23: 10–24.

    CAS  Article  Google Scholar 

  15. Sahoo S, Brickley DR, Kocherginsky M, Conzen SD . (2005). Coordinate expression of the PI3-kinase downstream effectors serum and glucocorticoid-induced kinase (SGK-1) and Akt-1 in human breast cancer. Eur J Cancer 41: 2754–2759.

    CAS  Article  Google Scholar 

  16. Shanmugam I, Cheng G, Terranova PF, Thrasher JB, Thomas CP, Li B . (2007). Serum/glucocorticoid-induced protein kinase-1 facilitates androgen receptor-dependent cell survival. Cell Death Differ 14: 2085–2094.

    CAS  Article  Google Scholar 

  17. Tu Y, Gardner A, Lichtenstein A . (2000). The phosphatidylinositol 3-kinase/AKT kinase pathway in multiple myeloma plasma cells: roles in cytokine-dependent survival and proliferative responses. Cancer Res 60: 6763–6770.

    CAS  PubMed  Google Scholar 

  18. van Zaanen HC, Lokhorst HM, Aarden LA, Rensink HJ, Warnaar SO, van der Lelie J et al. (1998). Chimaeric anti-interleukin 6 monoclonal antibodies in the treatment of advanced multiple myeloma: a phase I dose-escalating study. Br J Haematol 102: 783–790.

    CAS  Article  Google Scholar 

  19. Vasudevan KM, Barbie DA, Davies MA, Rabinovsky R, McNear CJ, Kim JJ et al. (2009). AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer. Cancer Cell 16: 21–32.

    CAS  Article  Google Scholar 

  20. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL . (1993). Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13: 2031–2040.

    CAS  Article  Google Scholar 

  21. Wu W, Chaudhuri S, Brickley DR, Pang D, Karrison T, Conzen SD . (2004). Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells. Cancer Res 64: 1757–1764.

    CAS  Article  Google Scholar 

  22. Zöllinger A, Stühmer T, Chatterjee M, Gattenlöhner S, Haralambieva E, Müller-Hermelink HK et al. (2008). Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma. Blood 112: 3403–3411.

    Article  Google Scholar 

Download references

Acknowledgements

We thank Brigitte Wollert-Wulf, Mandy Terne, Pia Herrmann (Berlin), Berit Størdal 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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to M Janz.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fagerli, UM., Ullrich, K., Stühmer, T. et al. 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. Oncogene 30, 3198–3206 (2011). https://doi.org/10.1038/onc.2011.79

Download citation

Keywords

  • multiple myeloma
  • cytokines
  • SGK1
  • STAT3

Further reading

Search

Quick links