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.
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).
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).
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.
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.
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.
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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.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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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
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