Molecular Cancer Research AM No Date
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Molecular Cancer Research 5, 271-281, March 1, 2007. doi: 10.1158/1541-7786.MCR-06-0274
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data, Komyod, et al.
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Komyod, W.
Right arrow Articles by Behrmann, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Komyod, W.
Right arrow Articles by Behrmann, I.

Signaling and Regulation

Constitutive Suppressor of Cytokine Signaling 3 Expression Confers a Growth Advantage to a Human Melanoma Cell Line

Waraporn Komyod1, Markus Böhm2, Dieter Metze2, Peter C. Heinrich1 and Iris Behrmann1,3

1 Institut für Biochemie, Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule Aachen, Aachen, Germany; 2 Department of Dermatology, University of Münster, Münster, Germany; and 3 Laboratoire de Biologie et Physiologie Intégrée, Université du Luxembourg, Luxembourg

Requests for reprints: Iris Behrmann, Laboratoire de Biologie et Physiologie Intégrée, Université du Luxembourg, 162A avenue de la Faïencerie, 1511 Luxembourg, Luxembourg. Phone: 352-46664-46740; Fax: 352-46664-46435. E-mail: iris.behrmann{at}uni.lu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, whereas late stage melanoma cells have often been reported to be "multi-cytokine–resistant." Here, we analyzed the melanoma cell line 1286, resistant towards the growth-inhibitory effects of interleukin 6 (IL-6), and oncostatin M (OSM), to better understand the mechanisms underlying cytokine resistance. Although the relevant receptors gp130 and OSMR are expressed at the cell surface of these cells, cytokine stimulation hardly led to the activation of Janus kinase 1 and signal transducer and activator of transcription (STAT)3 and STAT1. We found a high-level constitutive expression of suppressors of cytokine signaling 3 (SOCS3) that did not further increase after cytokine treatment. Importantly, upon suppression of SOCS3 by short interfering RNA, cells became susceptible towards OSM and IL-6: they showed an enhanced STAT3 phosphorylation and a dramatically increased STAT1 phosphorylation. Moreover, suppression of SOCS3 rendered 1286 cells sensitive to the antiproliferative action of IL-6 and OSM, but not of IFN-{alpha}. Interestingly, SOCS3–short interfering RNA treatment also increased the growth-inhibitory effect in cytokine-sensitive WM239 cells expressing SOCS3 in an inducible way. Thus, SOCS3 expression confers a growth advantage to these cell lines. Constitutive SOCS3 mRNA expression, although at lower levels than in 1286 cells, was found in nine additional human melanoma cell lines and in normal human melanocytes, although at the protein level, SOCS3 expression was marginal at best. However, in situ analysis of human melanoma specimens revealed SOCS3 immunoreactivity in 3 out of 10 samples, suggesting that in vivo SOCS3 may possibly play a role in IL-6 resistance in at least a fraction of tumors. (Mol Cancer Res 2007;5(2):271–81)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Many cytokines signal via Janus kinases (Jak) and signal transducers and activators of transcription (STAT) factors. Upon ligand-induced receptor clustering, tyrosine kinases of the Jak family associating with the cytoplasmic region of cytokine receptors become activated. They subsequently phosphorylate tyrosine residues of the receptor, which recruits other signaling proteins with matching SH2 domains such as STATs to the receptor. Upon phosphorylation of a single tyrosine residue, the STATs translocate as dimers to the nucleus, where they regulate the transcription of target genes (1, 2).

The Jak/STAT signaling pathway is subject to feedback inhibition by members of the suppressors of cytokine signaling (SOCS) family that are induced in a STAT-dependent fashion (3-5). They possess an SH2 domain and a COOH-terminal SOCS-box and inhibit Jak signaling upon binding to phosphorylated tyrosine residues of the cytokine receptors and/or to Janus kinases. Moreover, they are implicated in destabilizing their interacting partners because SOCS proteins can function as the substrate-recruiting component of ubiquitin ligases (6, 7). In several cancers, a dysregulation of SOCS expression has been observed. A tumor-suppressing function of SOCS1 and SOCS3 has been implied by reports on methylation-silenced SOCS genes in cancer cells (8-10). However, tumor cells constitutively expressing SOCS proteins may rather be indicative of a tumor-protecting function (11-15).

Cytokines play an important role in the growth regulation of melanoma cells. Although melanocytes and early stage melanoma cells are growth-inhibited by a variety of cytokines including interleukin 6 (IL-6), oncostatin M (OSM), or IFN-{gamma}, melanoma cells of advanced tumor stages are often found to be multi-cytokine–resistant (16-18). We have previously shown that STAT3 plays a key role in the IL-6- and OSM-mediated growth inhibition of A375 melanoma cells, whereas STAT1 plays a crucial role in growth inhibition mediated by IFN-{gamma} (19, 20). Cytokine resistance has also been associated with an impaired STAT3 activation (21). Moreover, lack of receptor expression can contribute to cytokine resistance (22), and it has recently been discovered that melanoma progression coincides with methylation silencing of the OSMRß gene (23). On the other hand, STAT3 has been shown to be constitutively active in melanoma cell lines and primary tumors, and inhibition of STAT activity induced apoptosis (24). Thus, STAT3 may play a complex role in melanoma.

In the present study, we analyzed a human melanoma cell line insensitive to IL-6 type cytokines with respect to the underlying mechanism of resistance. The cells constitutively express high levels of SOCS3 and our data indicate that SOCS3 expression confers a growth advantage to these cells.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
1286 Melanoma Cells Are Resistant to IL-6 and OSM and Constitutively Express SOCS3
While studying cytokine effects on melanoma cells, we noticed that the growth of 1286 cells was not inhibited by treatment with IL-6/sIL-6R complexes or OSM. However, the same cytokines dose-dependently inhibit the growth of A375 cells (Fig. 1A and B ; see also refs. 19, 25 and Supplementary Fig. S1). The relevant receptors gp130 as well as the OSMRß (but not the LIFR, see ref. 26) were present at the surface of 1286 and A375 cells (Fig. 1C), although the expression of the OSMR in 1286 cells was somewhat lower than in A375 cells.


Figure 1
View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 1. 1286 melanoma cells were resistant to cytokine-induced growth inhibition. A. Cells were cultured for 4 d in the presence of different concentrations of cytokines (initial density, 3,000 cells/well). Cells were treated with IL-6 in the presence of soluble IL-6R (1 µg/mL). Growth was assessed by an XTT test, values were recalculated according to untreated controls. Bars, SD of triplicate samples. B. Kinetics of growth inhibition. Two thousand cells per well were seeded into 96-well plates and cultured for different periods of time in the presence of IL-6/sIL-6R (100 ng/mL and 1 µg/mL, respectively), OSM (100 ng/mL), or with no cytokine added. Growth was assessed by an XTT test. Bars, SD of triplicate samples. C. 1286 cells express surface gp130 and OSMR. Cells were labeled using anti-gp130 monoclonal antibody, anti-OSMR monoclonal antibody, or anti-LIFR polyclonal antibody and the respective R-phycoerythrin–conjugated secondary antibodies (gray histograms). Cells treated with secondary antibody alone (white histograms).

 
IL-6 or OSM only induced a marginal STAT3 phosphorylation and no detectable STAT1 phosphorylation in 1286 (and in 888) cells (Fig. 2 ). In contrast, a prominent STAT3 and STAT1 phosphorylation was readily observed in A375 cells, WM9, and 729 cells (Fig. 2). 1286 cells show only a slight Jak1 phosphorylation after stimulation with OSM (Fig. 2C). Moreover, we observed no enhanced phosphorylation of the MAP kinases Erk1/2 or p38 (Fig. 2A). Most interestingly, when monitoring SOCS3 as a target gene of IL-6 type cytokines, we noticed that 1286 (but not 888) cells constitutively expressed SOCS3 (Fig. 2A-C). Cytokine treatment did not enhance SOCS3 expression in 1286 cells, in contrast to A375, WM9, and 729 cells, which showed a rapid and transient induction.


Figure 2
View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 2. Reduced Jak/STAT signal transduction in 1286 cells. A. Time-dependence of SOCS3 induction and STAT activation in melanoma cells. A375, WM9, 729, and 1286 melanoma cells were stimulated with OSM (20 ng/mL) for different periods of time, as indicated, or left untreated. Total cellular lysates were analyzed by Western blotting for the presence of phosphorylated STAT3, STAT1, p38, and Erks before reprobing of the blots with antibodies to STAT3, STAT1, p38, Erk1/2, and SOCS3. B. IL-6 and OSM hardly induce signaling events in 1286 melanoma cells. A375, WM9, 888, and 1286 melanoma cells were stimulated for 30 min with IL-6 (20 ng/mL) and sIL-6R (1 µg/mL) or with OSM (20 ng/mL) or left untreated. Total cellular lysates were analyzed by Western blotting for the presence of phosphorylated STAT3 and STAT1, before reprobing of the blots with antibodies to STAT3, STAT1, and SOCS3. C. Reduced Jak1 phosphorylation in 1286 cells. WM9, 729, and 1286 melanoma cells were stimulated with OSM (10 ng/mL) for different periods of time, as indicated, or left untreated. Jak1 was immunoprecipitated from total cellular lysates, phosphorylation was analyzed by staining the Western blot with PY99 before reprobing the blot with Jak1 antibody. Aliquots of the lysates were additionally analyzed by Western blotting for the presence of Jak1 and phosphorylated STAT3 before reprobing of the latter blot with antibodies to STAT3 and SOCS3.

 
Taken together, 1286 cells have constitutively high levels of SOCS3 and essentially do not respond to IL-6 type cytokines, neither by activation of the Jak/STAT pathway nor by growth inhibition.

Suppression of SOCS3 Expression Breaks Cytokine Resistance of 1286 Cells
To better understand the correlation between SOCS3 expression and cytokine resistance, we suppressed SOCS3 protein expression by a short interfering RNA (siRNA) approach. Transfection of 1286 cells with SOCS3 siRNA almost completely abrogated SOCS3 mRNA expression as measured by reverse transcription-PCR (RT-PCR; Fig. 3A ). Moreover, constitutive SOCS3 protein expression was significantly reduced (Fig. 3B). SOCS3 suppression slightly (10-20%, median fluorescence channel) increased the surface expression of both gp130 and OSMR, but not of LIFR (data not shown).


Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 3. Suppression of SOCS3 in 1286 cells leads to an enhanced sensitivity towards IL-6. A. SOCS3 expression was suppressed using siRNA transfected with jetSI ENDO transfection reagent. Knockdown efficiency at the mRNA level was verified by RT-PCR after 24 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CIS, cytokine-inducible SH2-containing protein. B. Suppression of SOCS3 expression by SOCS3 siRNA increases STAT1 and STAT3 phosphorylation in 1286 cells. 1286 cells were transfected with 50 nmol/L of SOCS3 siRNA or control siRNA for 24 h and then stimulated for 15 min with IL-6 (20 ng/mL) and sIL-6R (1 µg/mL) or with OSM (10 ng/mL) or left untreated. Total cellular lysates were analyzed by Western blotting for the presence of phosphorylated STAT3 and STAT1 before reprobing of the blots with antibodies to STAT3, STAT1, p38, and SOCS3. C. 1286 melanoma cells were transfected with 50 nmol/L of SOCS3 siRNA or control siRNA for 24 h and then stimulated for 15 min with IL-6 (20 ng/mL) and sIL-6R (1 µg/mL) or left untreated. Nuclear extracts were analyzed by electrophoretic mobility shift assay for binding activities to the SIE probe. D. 1286 cells were transfected with 50 nmol/L of SOCS3 siRNA or control siRNA. Twenty-four hours after transfection, cells were cultured for 3 d in the presence of different concentrations of IL-6 in combination with sIL-6R (1 µg/mL) or with no cytokine added. Growth was assessed by an XTT test, values were recalculated according to untreated controls. Bars, SD of triplicate samples. E. 1286 cells were transfected with 50 nmol/L of SOCS3 siRNA or control siRNA for 24 h. Two thousand cells per well were seeded into 96-well plates and cultured for different periods of time in the presence of IL-6/sIL-6R (100 ng/mL and 1 µg/mL, respectively), OSM (100 ng/mL), or with no cytokine added. Growth was assessed by an XTT test. Bars, SD of triplicate samples. F. SOCS3 suppression of the cells used in (E) was still evident in Western blot analysis after 4 d of culture.

 
We investigated signaling events in 1286 cells treated with SOCS3 siRNA or with scrambled control siRNA. A slight increase in IL-6- and OSM-mediated STAT3 phosphorylation was observed in SOCS3-suppressed cells. However, more strikingly, we consistently observed a very strong STAT1 phosphorylation upon treatment with IL-6 and OSM, compared with cells that had been treated with control siRNA or that remained untransfected (Fig. 3B). The increased phosphorylation of STAT1 upon SOCS3 suppression correlated with a strong STAT1 band in the gel shift assay (Fig. 3C).

We then investigated whether down-regulation of SOCS3 expression would break the resistance of these cells towards the growth inhibitory effect of cytokines. Twenty-four hours after siRNA transfection, cells were treated with IL-6 in the presence of soluble IL-6R, and cell growth was assessed 3 days later by a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Fig. 3D). Strikingly, SOCS3 suppression increased the growth-inhibitory effect significantly: cells treated with control siRNA were not affected by the cytokine, as shown previously for the untransfected cells (see Fig. 1A). In contrast, cells treated with SOCS3 siRNA were dose-dependently inhibited by IL-6 treatment (Fig. 3D). Similarly, upon SOCS3 suppression, the cytokine sensitivity was also evident in the time course experiment shown in Fig. 3E and F. We consistently observed values between 60% and 80% of growth compared with untreated control cells. We often also noted that SOCS3-suppressed cells grew slower than control siRNA–treated cells, even when no cytokine was added (see Fig. 3E). Two other siRNA oligonucleotides were less effective as the one used in the experiments shown here. Interestingly, their reduced effect on SOCS3 suppression was paralleled by a weaker "restoration" of STAT phosphorylation and growth inhibition, indicating that SOCS3 confers cytokine resistance in a dose-dependent manner (Supplementary Fig. S2).

Suppression of Inducible SOCS3 Expression Increases the Growth-Inhibitory Effect of IL-6 Type Cytokines in Sensitive WM239 Cells
To address the question of whether SOCS3 suppression could also enhance cytokine responsiveness in sensitive melanoma cells, WM239 cells (see Supplementary Fig. S1) were transfected with SOCS3 siRNA or with control siRNA. These cells strongly express SOCS3 after cytokine treatment (Fig. 4A ). SOCS3 siRNA reduced inducible SOCS3 expression. As previously observed in 1286 cells (Fig. 3B and C), SOCS3 suppression coincided with prominent STAT1 phosphorylation (Fig. 4A). Interestingly, suppression of inducible SOCS3 enhanced the growth-inhibitory effect of IL-6/sIL-6R and of OSM. Even without cytokine treatment, control cells grew better than SOCS3-suppressed cells (Fig. 4B). Thus, SOCS3 expression also promotes growth in sensitive melanoma cells.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 4. Suppression of inducible SOCS3 expression increases cytokine sensitivity of WM239 cells. A. WM239 cells were transfected with 50 nmol/L of SOCS3-siRNA or control siRNA for 24 h and then stimulated for 60 min with IL-6 (20 ng/mL) and sIL-6R (1 µg/mL), or with 20 ng/mL OSM or left untreated. Total cellular lysates were analyzed by Western blotting for the presence of phosphorylated STAT1 and STAT3 before reprobing of the blots with antibodies to STAT1, STAT3, Erk1/2, and SOCS3. B. WM239 cells were transfected with 50 nmol/L of SOCS3-siRNA or control-siRNA. Twenty-four hours after transfection, 2,000 cells/well were seeded into 96-well plates and cultured for different periods of time in the presence of IL-6/sIL-6R (100 ng/mL and 1 µg/mL, respectively), OSM (100 ng/mL), or with no cytokine added. Growth was assessed by an XTT test. Bars, SD of triplicate samples.

 
SOCS3 Suppression Does Not Break the Resistance of 1286 Cells against IFN-{alpha}
Type 1 IFNs play an important role in adjuvant melanoma therapy but only 20% of the patients benefit from a treatment. The reasons for resistance in most patients are not well understood. Therefore, we investigated whether SOCS3 expression in 1286 cells might also contribute to their resistance against IFN-{alpha} (see Supplementary Fig. S1). However, the suppression of SOCS3 expression did not sensitize 1286 cells against the growth-inhibitory effect of IFN-{alpha} (Fig. 5 ) and did not enhance IFN-{alpha}–mediated STAT1 phosphorylation (data not shown). This indicates that constitutive SOCS3 is not the reason for IFN-{alpha} resistance of these cells.


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 5. Suppression of SOCS3 does not break the resistance of 1286 cells against IFN-{alpha}. 1286 cells were transfected with 50 nmol/L of SOCS3 siRNA or control siRNA. Twenty-four hours after transfection, 2,000 cells/well were seeded into 96-well plates and cultured for different periods of time in the presence of IL-6/sIL-6R (100 ng/mL and 1 µg/mL, respectively), IFN-{alpha} (5,000 units/mL), or with no cytokine added. Growth was assessed by an XTT test. Bars, SD of triplicate samples. SOCS3 suppression of the cells used for proliferation assay was still evident in Western blot analysis after 4 d of culture.

 
Enhanced SOCS3 Expression Is Not a General Phenomenon of Melanoma Cells In vitro
To further assess whether a constitutive SOCS3 expression is frequently observed in melanoma, we analyzed nine additional melanoma cells and primary melanocytes (normal human epidermal melanocytes). As shown in Fig. 6 , 1286 cells clearly expressed the highest levels of SOCS3 protein. For A375, WM9, and 888 cells we sometimes, but not consistently, observed a faint signal in the Western blot (see also Fig. 2). PCR data confirmed highest mRNA expression in 1286 cells (Fig. 6B and C). Compared with melanocytes, 1286 cells express nine times more SOCS3 mRNA. Other cell lines, including A375 and WM9, showed a 2- to 3-fold increase in SOCS3 expression. Three cell lines expressed lower SOCS3 mRNA levels than melanocytes. It should be noted that SOCS3 mRNA expression did not correlate well with cytokine sensitivity (see Supplementary Fig. S1). Taken together, 1286 cells show the highest expression levels of SOCS3, both on protein and on mRNA levels. However, such a high expression does not seem to be a general phenomenon of melanoma cells.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 6. A. Highest expression of SOCS3 protein in 1286 melanoma cells. B. Analysis of constitutive expression of SOCS3 by RT-PCR. C. The relative SOCS3 mRNA levels were determined in cDNA samples by quantitative real-time PCR as described in Materials and Methods. The indicated relative gene expression shows expression levels that were normalized to ß-actin expression as a standard. Expression levels were determined in three independent experiments. Columns, mean; bars, SD.

 
SOCS3 Immunoreactivity in Human Melanoma In situ
To further investigate if SOCS3 is expressed in melanoma cells in situ, and thus, could mediate IL-6 resistance in vivo, we did immunohistochemical studies on a limited number of human melanoma specimens (n = 10) derived from early and advanced primary cutaneous melanomas (n = 8) as well as melanoma metastases (n = 2). SOCS3 immunoreactivity was detectable in three primary cutaneous melanomas (Clark levels II, III, and IV) as a fine granular cytoplasmic staining of the tumor cells (Fig. 7A and C ). The detected SOCS3 immunostaining was not uniformly present in all tumor cells but was confined to cell clones, sometimes located at the periphery of the tumor. In contrast, sections incubated with isotype control IgG did not reveal any immunostaining (Fig. 7B and D). In 4 out of 10 samples, some SOCS3 immunoreactivity was also detectable in basal epidermal keratinocytes, and in 8 out of 10 samples, it was also in selected skin appendages, i.e., in secretary epithelia of eccrine glands and in hair follicle epithelia (data not shown), suggesting that SOCS3 expression is not a melanoma-specific marker.


Figure 7
View larger version (152K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 7. Detection of SOCS3 immunoreactivity in human melanoma in situ. Deparaffinized tissue sections were stained with a polyclonal anti-SOCS3 antibody and bound antibodies were visualized by the immunoperoxidase technique. A. SOCS3 immunostaining of a primary cutaneous melanoma (Clark level III). Note intense staining of some tumor cells but absence of staining of others (original magnification, x200). B. Negative control of (A; original magnification, x100). C. Primary cutaneous melanoma (Clark level IV). Note the SOCS3 immunoreactive tumor cell nests, whereas other tumor cells remain negative. The brown staining in contrast is due to melanin (original magnification, x200). D. Negative control of (C; original magnification, x100).

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The major findings of this study are (a) cytokine-resistant 1286 melanoma cells show constitutive expression of SOCS3 and (b) SOCS3 expression confers a growth advantage to the cells as suppression of SOCS3 restores or increases sensitivity against IL-6 type cytokines.

IL-6 type cytokines lead to STAT3 and STAT1 tyrosine phosphorylation, although the latter is generally only observed on short-term treatment with a high dose of IL-6 (19, 27). For A375 cells, we have previously shown that STAT3 is crucial for IL-6- and OSM-mediated growth inhibition (19). The signal transduction of IL-6 type cytokines is subject to feedback inhibition by SOCS proteins, with SOCS3 playing the most important role (7). Overexpression of SOCS3 shuts off cytokine signaling: A375 cells heterologously expressing SOCS3 are resistant to the growth-inhibitory action of OSM (28). These findings are in full accordance with the results of our present study.

The "quality" of a STAT response can be modulated by the presence or absence of other proteins, as first described for cells lacking STAT3: in the absence of STAT3, IL-6 mediates a STAT1-dominated IFN-{gamma}–like response (29). Interestingly, suppression of SOCS3 in 1286 cells modulated the STAT response not only quantitatively but also in a qualitative manner by drastically increasing the STAT1 response to IL-6 (Fig. 3). It has previously been described that SOCS3 can influence the pattern of STAT activation: in the absence of SOCS3, it was observed that macrophages, hepatocytes, and murine embryonic fibroblasts respond to IL-6 type cytokines with a prolonged STAT1 activation (in addition to a prolonged STAT3 activation; refs. 30-32), which could lead to the expression of IFN-{gamma}-responsive genes (31, 32). An IFN-{gamma}–like response in the absence of SOCS3 was also discussed to be the cause for changing the phenotype of B7-stimulated dendritic cells from immunostimulatory to immunosuppressive (33). For A375 melanoma cells, we have previously shown that STAT1 activation can mediate growth inhibition (20). It is therefore likely that STAT1 contributes to IL-6–mediated growth inhibition in SOCS3-suppressed 1286 cells. However, IL-6 does not elicit a full-blown IFN-{gamma}–like response in SOCS3-suppressed 1286 cells, e.g., we did not observe an up-regulation of MHC class I expression, a hallmark of an IFN-{gamma} response (data not shown). Taken together, our data underline the role of SOCS3 not only in limiting the extent of a STAT response quantitatively but also in the "sculpting" of the STAT response in a qualitative way.

The expression of SOCS3 was previously described to be altered in cancer cells and either tumor-suppressing or tumor-protecting functions have been discussed. The SOCS3 gene was found to be silenced by promoter methylation in lung cancer, breast cancer, and mesothelioma cells as well as in primary lung cancer tissue samples (8), in hepatocellular carcinoma (9), and in squamous cell carcinoma of the head and neck (10). Restoration of SOCS3 suppressed growth and resulted in apoptosis (8-10). Although SOCS3 protein was virtually undetectable in most melanoma cells we tested prior to cytokine treatment, the SOCS3 gene is probably not silenced in melanoma cells, as the SOCS3 message could readily be detected, albeit at varying levels, in all melanoma cells as in melanocytes (Fig. 6).

In other cancers such as anaplastic large cell lymphoma, SOCS3 is overexpressed (34). Constitutive SOCS3 expression in T cell lymphoma depended on STAT3 signaling because expression of a dominant negative version of STAT3 inhibited SOCS3 expression (11, 12). Constitutively expressed SOCS3 was shown to protect chronic myeloid leukemia cells against IFN-{alpha} treatment (13, 14). Thus, for SOCS3-expressing tumors, SOCS3 may not be a tumor suppressor but rather a protector, which may be of special relevance in tumors that are treated with type 1 IFNs. However, SOCS3 expression selectively mediates the resistance of 1286 melanoma cells against IL-6 type cytokines but does not underlie their resistance to IFN-{alpha} (Fig. 5).

The suppression of constitutive SOCS3 expression slightly increased the surface expression of both gp130 and OSMR (data not shown). SOCS proteins as ubiquitin ligase components are known to affect the half-life of interacting proteins. Further studies will show whether this receptor up-regulation could contribute to cytokine sensitivity. Interestingly, in a recent study, loss of OSMR expression correlated with melanoma progression (23).

In contrast to T cell lymphoma cells (12), dominant negative STAT3 (STAT3F) did not affect SOCS3 expression in 1286 cells (Supplementary Fig. S3). We observed a down-regulation of SOCS3 expression when treating cells with a N-methyl-2-deoxyadenosine/adenosine/D,L-homocysteine inhibitor cocktail (see Supplementary Fig. S4) which affects multiple signaling pathways (35). However, more specific inhibitors of MEK and p38 did not influence SOCS3 expression (Supplementary Fig. S4), although in other situations, these signaling pathways have been implicated in the regulation of SOCS3 expression (36-38). The cDNA sequence of SOCS3 of 1286 cells shows no mutations (data not shown). Furthermore, we do not have evidence for tyrosine phosphorylation of the SOCS3 protein or for a prolonged half-life of the protein or mRNA (data not shown). Thus, the molecular basis for constitutive SOCS3 expression in 1286 cells remains to be elucidated.

Interestingly, SOCS1, another member of the SOCS family implicated mainly in IFN signaling (for a review, see ref. 39), was found to be constitutively expressed in melanoma cells (15). In this report, SOCS1 expression did not correlate with IL-6 sensitivity, but a potential involvement in growth promotion and/or IFN resistance of melanoma cells was discussed based on the detected SOCS1 immunoreactivity in situ, which increased as a function of the stage of disease (15). With regard to SOCS1 expression, melanoma cells are thus different from other cancer cells in which the SOCS1 gene is silenced by methylation, such as in pancreatic cancer (40, 41), in gastric cancer (42), in human hepatoblastomas (43, 44), and in hematopoietic malignancies (45-48). Our present analysis of 10 human melanomas indicates that SOCS3 immunoreactivity is likewise present in a portion of tumors. Further studies on a higher number of melanoma specimens are currently under way to precisely clarify whether SOCS3 behaves as a progression marker of melanoma and/or whether its in situ expression (alone or in combination with SOCS1) might correlate with IFN-{alpha} responsiveness in vivo.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture and Reagents
Normal human epidermal melanocytes were purchased from Cambrex Bio Science (Verviers, Belgium) and cultured in MBM-4 medium with the recommended supplements and growth factors. Human A375 melanoma cells were purchased from American Type Culture Collection (Manassas, VA). WM239 and WM9 cells (49) were received from Dr. R.S. Kerbel (Sunnybrook Health Science Centre, Toronto, Canada). Cell lines 586, 729, 888, 1102, 1286, and 1287 were kindly provided by Dr. Marcin Kortylewski (City of Hope National Medical Center, Duarte, CA). Mel Im cells were obtained from Dr. Anja Bosserhoff (University of Regensburg, Germany). Cells were maintained in RPMI 1640 (Gibco Life Technologies, Karlsruhe, Germany) supplemented with 5% FCS (Invitrogen, Karlsruhe, Germany), 100 mg/L of streptomycin, and 60 mg/L of penicillin. Cells were grown at 37°C in a water-saturated atmosphere at 5% CO2.

Human recombinant IFN-{gamma} and OSM were purchased from Peprotech (London, United Kingdom). Recombinant IL-6 and soluble IL-6 receptor were prepared as described previously (50, 51). Adenosine, D,L-homocysteine, N-methyl-2-deoxyadenosine and 5'-methylthioadenosine were purchased from Sigma (Taufkirchen, Germany) and dissolved in culture medium. SB 201290 and U0126 were purchased from Calbiochem (Darmstadt, Germany) and dissolved in DMSO.

Generation of Transfectants
The wild-type and dominant negative STAT constructs in the pCAGGS plasmid were gifts from Koichi Nakajima and Toshio Hirano (Osaka, Japan). For preparation of stable transfectants, 2 x 106 cells were transfected with 5 µg of the respective plasmid DNA using FuGENE 6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions. Transfectants were selected in the presence of 1 mg/mL of G418 (Sigma). Heterologous expression of STATs was assessed with a monoclonal antibody directed against the hemagglutinin-Tag from Cell Signaling Technology, Inc. (Frankfurt, Germany).

Growth Inhibition Assay
Viable cells (3 x 103) were seeded in triplicate into 96-microwell plates and incubated with various concentrations of cytokines in medium containing 1% FCS. After 4 days of culture, an XTT colorimetric assay (Roche, Mannheim, Germany) was done as described previously (19). The percentage of growth inhibition was calculated in relation to the growth of untreated control cells. For the experiments on time-dependence, 1.5 x 103 viable cells/well were seeded with or without cytokines and XTT tests were done after different periods of incubation.

Flow Cytometry
Cells were resuspended in cold PBS supplemented with 5% FCS and 0.1% sodium azide (PBS/azide). Cells (5 x 105 to 1 x 106) in 100 µL of PBS/azide were incubated with 1 µg/mL monoclonal anti–MHC-I (Sigma), anti-OSMR (Santa Cruz Biotechnology, Heidelburg, Germany), or anti-gp130 (BR3; gifts from Dr. J. Wijdenes, Besançon, France) or polyclonal anti-LIFR (Santa Cruz Biotechnology) for 30 min at 4°C. Cells were then washed with cold PBS/azide and subsequently incubated in darkness with a 1:100 dilution of respective R-phycoerythrin–conjugated secondary antibodies (Dianova, Hamburg, Germany) for 30 min at 4°C. Cells were again washed with cold PBS/azide, and then 104 cells per sample were analyzed by flow cytometry using a FACScalibur (Becton Dickinson, Heidelburg, Germany) equipped with a 488 nm argon laser.

Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared according to Wegenka et al. (52), and protein concentrations were measured using a Bio-Rad (München, Germany) protein assay. Briefly, 10 fmol (10.000 cpm) of a {alpha}-32P–labeled double-stranded oligonucleotide was added to nuclear extracts containing 5 µg of protein and incubated in gel shift incubation buffer: 10 mmol/L of HEPES (pH 7.8), 1 mmol/L of EDTA, 5 mmol/L of MgCl2, 10% glycerol, 5 µmol/L of DTT, 0.7 µmol/L of phenylmethylsulfonyl fluoride, 0.1 mg/mL of poly(dI-dC), and 1 mg/mL of bovine serum albumin for 10 min at room temperature. The protein-DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in 0.25-fold Tris-borate EDTA at 20 V/cm for 4 h. Gels were fixed in a water solution of 10% methanol and 10% acetic acid for 30 min, dried, and autoradiographed. Data were further analyzed using a Personal FX PhosphorImager with the Quantity One software (Bio-Rad). Electrophoretic mobility shift assays were done using the m67SIE probe (m67SIE: 5'-GAT CCG GGA GGG ATT TAC GGG AAA TGC TG-3') which binds to STAT1 and STAT3.

Western Blot Analysis and Antibodies
Cells were lysed on the plate in lysis buffer containing 20 mmol/L of HEPES (pH 7.4), 1% Triton X-100, 100 mmol/L of NaCl, 50 mmol/L of NaF, 10 mmol/L of ß-glycerophosphate, 1 mmol/L of sodium vanadate, 1 mmol/L of phenylmethylsulfonyl fluoride, 1 mmol/L of benzamidine, 5 µg/mL of aprotinin, 3 µg/mL of pepstatin, and 5 µg/mL of leupeptin. Lysates were clarified at 13,000 rpm for 10 min at 4°C and protein concentration in the supernatant was determined with the Bio-Rad protein assay. Equal amounts of total cellular proteins were separated by SDS-PAGE on 12% or 7.5% gels, transferred to a polyvinylidene difluoride membrane (PALL, Germany) and probed with the respective antibodies. Rabbit anti–phospho-STAT1 (Tyr701), rabbit anti–phospho-STAT3 (Tyr705), mouse anti–phospho-Erk1/2, mouse anti–hemagglutinin-Tag were from Cell Signaling Technology. Mouse anti-STAT1 and mouse anti-STAT3 were purchased from Transduction Laboratories. Rabbit anti-Jak1, mouse anti-pTyr (PY99), rabbit anti-p38, goat anti-Erk1, goat anti-Erk2, and goat anti-SOCS3 were from Santa Cruz Biotechnology. Rabbit anti-SOCS3 was purchased from IBL (Hamburg, Germany). Rabbit anti–phospho-p38 was obtained from Promega (Mannheim, Germany). The horseradish peroxidase–conjugated secondary antibodies were purchased from Dako (Hamburg, Germany). Signals were detected using the enhanced chemiluminescence system (Amersham Biosciences, Freiburg, Germany). Before reprobing, blots were stripped in 2% SDS, 100 mmol/L of ß-mercaptoethanol in 62.5 mmol/L Tris-HCl (pH 6.7), for 20 min at 75°C.

SiRNA Synthesis and Transfection
All siRNAs used in this study were synthesized by Eurogentec (Seraing, Belgium) and received as desalted, preannealed duplexes in desalted-purified formats. The siRNA sequences used for targeting human SOCS3 were sense, 5'-CCAAGAACCUGCGCAUCCAdTdT-3'; antisense, 5'-UGGAUGCGCAGGUUCUUG GdTdT-3' (most-effective siRNA3). Control siRNAs (see Supplementary Fig. S1) were siRNA1 sense: 5'-AGAGCCUAUUACAUCUACUdTdT-3'; siRNA1 antisense, 5'-AGUAGAUGUAAUAGGCUCUdTdT-3'; siRNA2 sense, 5'-AGACCCAGUCUGGGACCAAdTdT-3'; siRNA2 antisense, 5'-UUGGUCCCAGACUGGGUCUdTdT-3'. A nonspecific siRNA, scrambled negative control siRNA was used as a control. Transient transfection of siRNA oligonucleotides was done at 50 nmol/L with jetSI-ENDO Transfection Reagent (Eurogentec) according to the manufacturer's protocol.

Preparation of RNA, RT-PCR Analysis, and Sequencing
Total RNA was isolated from melanoma cell lines using the RNeasy Mini kit from Qiagen (Hilden, Germany) as described by the manufacturer. Expression of SOCS transcripts was determined by RT-PCR analysis using specific primers as described previously (53). RT-PCR was done with 1 µg of total RNA using the One-Step RT-PCR kit from Qiagen. The amplification program consisted of one cycle at 50°C for 30 min, 95°C for 15 min, followed by 35 cycles at 94°C for 40 s, 58°C for 30 s, 72°C for 30 s with a final extension at 72°C for 10 min. PCR products were separated electrophoretically on 1.5% TAE agarose gels and visualized by ethidium bromide staining.

For sequencing, the cDNA of human SOCS3 was synthesized from RNA by RT-PCR using specific primers: SOCS3 forward 5'-CTGGCTCCGTGCGCCATG-3' and SOCS3 reverse 5'-GGAAGCTGAGGAATTGAAGGAGAA-3', resulting in a 1,013 bp fragment. RT-PCR products were gel-purified using a gel extraction kit (Qiagen) and cloned into pCR2.1-TOPO vector (Invitrogen) and sequenced.

Quantitative Real-time PCR
Real-time PCR was done on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Darmstadt, Germany). The primers for human SOCS3 and ß-actin were obtained from Eurogentec. Relative quantification of SOCS3 expression was carried out using SYBR Green PCR reagents from PE Applied Biosystems (Darmstadt, Germany). Briefly, 1 µg of purified RNA was reverse-transcribed using the First Strand cDNA Synthesis Kit for RT-PCR (AMV; Roche) with random hexamers primers according to the manufacturer's description. The cDNAs for SOCS3 and internal control ß-actin were amplified using specific primers for SOCS3 (forward primer, 5'-CACCTGGACTCCTATGAGAAAGTCA-3'; and reverse primer, 5'-GGGGCATCGTACTGGTCCAGGAA-3') or for ß-actin (forward primer, 5'-CCCTGAGGCACTCTTCCAG-3'; and reverse primer, 5'-TGCCACAGGACTCCATGCCC-3'). The PCR reaction was done in a final volume of 25 µL containing 2.5 µL of cDNA, 100 nmol/L of each primer, 12.5 µL of SYBR Green PCR buffer (Applied Biosystems). After denaturing for 15 min at 94°C, amplification was done by 40 cycles of 15 s at 94°C, and a combined annealing/extension step of 60 s at 60°C using the ABI prism 7000 (Applied Biosystems). SOCS3 and ß-actin were amplified independently in separate reaction wells in triplicate. The real-time PCR efficiencies were determined for each primer/probe set from standard curves generated from serial dilutions of a cDNA sample of OSM-stimulated human melanoma A375 cells. The quantification of gene expression was calculated using a mathematical model including the PCR efficiencies (54). The level of SOCS3 gene expression in the test samples was normalized to the corresponding ß-actin level and is reported as the fold difference.

Immunohistochemistry
Human melanoma specimens (n = 10) derived from patients undergoing routine surgery for therapeutic or diagnostic reasons were examined. Analysis and processing of these samples was previously approved by the ethical committee of the University of Münster, Münster, Germany. The specimens included early primary cutaneous melanomas with Clark level II (n = 2), Clark level III (n = 3), Clark level IV (n = 2), Spitzoid melanoma (n = 1, Clark level IV), and melanoma metastases (n = 2). Specimens were fixed in 7% buffered paraformaldehyde, dehydrated, embedded in paraffin, and mounted on Tissue-Tek (Mikrom, Walldorf, Germany). Paraffin-embedded sections were subsequently deparaffinized by routine methods followed by epitope unmasking with proteinase K (Dako, Hamburg, Germany) for 10 min at room temperature in a wet chamber. After washing with PBS, sections were stained at 1:100 with a polyclonal antibody against SOCS3 (IBL) for 45 min. Negative controls consisted of the isotype control IgG incubated at the same protein concentration as the primary antibody. After washing, sections were further processed for immunoperoxidase staining using the Polylink system from DCS (Hamburg, Germany) and AEC as substrate. Sections were finally counterstained with Mayer's hematoxylin (Merck, Darmstadt, Germany). Immunostaining steps were semiautomatically processed by the Autostainer 480 (Mikrom).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Drs. Serge Haan and Stephanie Kreis for critical reading of the manuscript. We are grateful to Dr. Alexandra Dreuw for help in qPCR calculations and to Dr. Ulrike Sommer for support in detecting SOCS3 phosphorylation. We also thank Drs. Toshio Hirano and Koichi Nakajima for the expression plasmid encoding dominant negative STAT3.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Fonds der Chemischen Industrie (Frankfurt am Main) and the Deutsche Forschungsgemeinschaft (Bonn), Be1919/5-2.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

Received 8/25/06; revised 12/22/06; accepted 1/23/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 2003;374:1–20.[CrossRef][Medline]
  2. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem 1998;67:227–64.[CrossRef][Medline]
  3. Starr R, Willson TA, Viney EM, et al. A family of cytokine-inducible inhibitors of signalling. Nature 1997;387:917–21.[CrossRef][Medline]
  4. Endo TA, Masuhara M, Yokouchi M, et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 1997;387:921–4.[CrossRef][Medline]
  5. Naka T, Narazaki M, Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 1997;387:924–9.[CrossRef][Medline]
  6. Johnston JA. Are SOCS suppressors, regulators, and degraders? J Leukoc Biol 2004;75:743–8.[Abstract/Free Full Text]
  7. Wormald S, Hilton DJ. Inhibitors of cytokine signal transduction. J Biol Chem 2004;279:821–4.[Abstract/Free Full Text]
  8. He B, You L, Uematsu K, et al. SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc Natl Acad Sci U S A 2003;100:14133–8.[Abstract/Free Full Text]
  9. Niwa Y, Kanda H, Shikauchi Y, et al. Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene 2005.
  10. Weber A, Hengge UR, Bardenheuer W, et al. SOCS-3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions and causes growth inhibition. Oncogene 2005;24:6699–708.[CrossRef][Medline]
  11. Brender C, Nielsen M, Kaltoft K, et al. STAT3-mediated constitutive expression of SOCS-3 in cutaneous T-cell lymphoma. Blood 2001;97:1056–62.[Abstract/Free Full Text]
  12. Brender C, Lovato P, Sommer VH, et al. Constitutive SOCS-3 expression protects T-cell lymphoma against growth inhibition by IFN{alpha}. Leukemia 2005;19:209–13.[CrossRef][Medline]
  13. Sakai I, Takeuchi K, Yamauchi H, Narumi H, Fujita S. Constitutive expression of SOCS3 confers resistance to IFN-{alpha} in chronic myelogenous leukemia cells. Blood 2002;100:2926–31.[Abstract/Free Full Text]
  14. Takeuchi K, Sakai I, Narumi H, et al. Expression of SOCS3 mRNA in bone marrow cells from CML patients associated with cytogenetic response to IFN-{alpha}. Leuk Res 2005;29:173–8.[CrossRef][Medline]
  15. Li Z, Metze D, Nashan D, et al. Expression of SOCS-1, suppressor of cytokine signalling-1, in human melanoma. J Invest Dermatol 2004;123:737–45.[CrossRef][Medline]
  16. Swope VB, Abdel-Malek Z, Kassem LM, Nordlund JJ. Interleukins 1{alpha} and 6 and tumor necrosis factor-{alpha} are paracrine inhibitors of human melanocyte proliferation and melanogenesis. J Invest Dermatol 1991;96:180–5.[CrossRef][Medline]
  17. Lu C, Vickers MF, Kerbel RS. Interleukin 6: a fibroblast-derived growth inhibitor of human melanoma cells from early but not advanced stages of tumor progression. Proc Natl Acad Sci U S A 1992;89:9215–9.[Abstract/Free Full Text]
  18. Lu C, Rak JW, Kobayashi H, Kerbel RS. Increased resistance to oncostatin M-induced growth inhibition of human melanoma cell lines derived from advanced-stage lesions. Cancer Res 1993;53:2708–11.[Abstract/Free Full Text]
  19. Kortylewski M, Heinrich PC, Mackiewicz A, et al. Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1. Oncogene 1999;18:3742–53.[CrossRef][Medline]
  20. Kortylewski M, Komyod W, Kauffmann ME, Bosserhoff A, Heinrich PC, Behrmann I. Interferon-{gamma}-mediated growth regulation of melanoma cells: involvement of STAT1-dependent and STAT1-independent signals. J Invest Dermatol 2004;122:414–22.[CrossRef][Medline]
  21. Böhm M, Schulte U, Funk JO, et al. Interleukin-6-resistant melanoma cells exhibit reduced activation of STAT3 and lack of inhibition of cyclin E-associated kinase activity. J Invest Dermatol 2001;117:132–40.[CrossRef][Medline]
  22. Oh JW, Katz A, Harroch S, Eisenbach L, Revel M, Chebath J. Unmasking by soluble IL-6 receptor of IL-6 effect on metastatic melanoma: growth inhibition and differentiation of B16-F10.9 tumor cells. Oncogene 1997;15:569–77.[CrossRef][Medline]
  23. Lacreusette A, Nguyen JM, Pandolfino MC, et al. Loss of oncostatin M receptor ß in metastatic melanoma cells. Oncogene 2006.
  24. Niu G, Bowman T, Huang M, et al. Roles of activated Src and Stat3 signaling in melanoma tumor cell growth. Oncogene 2002;21:7001–10.[CrossRef][Medline]
  25. Zarling JM, Shoyab M, Marquardt H, Hanson MB, Lioubin MN, Todaro GJ. Oncostatin M: a growth regulator produced by differentiated histiocytic lymphoma cells. Proc Natl Acad Sci U S A 1986;83:9739–43.[Abstract/Free Full Text]
  26. Auguste P, Guillet C, Fourcin M, et al. Signaling of type II oncostatin M receptor. J Biol Chem 1997;272:15760–4.[Abstract/Free Full Text]
  27. Haan S, Keller JF, Behrmann I, Heinrich PC, Haan C. Multiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation. Cell Signal 2005;17:1542–50.[CrossRef][Medline]
  28. Magrangeas F, Boisteau O, Denis S, Jacques Y, Minvielle S. Negative regulation of oncostatin M signaling by suppressor of cytokine signaling (SOCS-3). Eur Cytokine Netw 2001;12:309–15.[Medline]
  29. Costa-Pereira AP, Tininini S, Strobl B, et al. Mutational switch of an IL-6 response to an interferon-{gamma}-like response. Proc Natl Acad Sci U S A 2002;99:8043–7.[Abstract/Free Full Text]
  30. Lu Y, Fukuyama S, Yoshida R, et al. Loss of SOCS3 gene expression converts STAT3 function from anti-apoptotic to pro-apoptotic. J Biol Chem 2006;281:36683–90.[Abstract/Free Full Text]
  31. Croker BA, Krebs DL, Zhang JG, et al. SOCS3 negatively regulates IL-6 signaling in vivo. Nat Immunol 2003;4:540–5.[CrossRef][Medline]
  32. Lang R, Pauleau AL, Parganas E, et al. SOCS3 regulates the plasticity of gp130 signaling. Nat Immunol 2003;4:546–50.[CrossRef][Medline]
  33. Orabona C, Belladonna ML, Vacca C, et al. Cutting edge: silencing suppressor of cytokine signaling 3 expression in dendritic cells turns CD28-Ig from immune adjuvant to suppressant. J Immunol 2005;174:6582–6.[Abstract/Free Full Text]
  34. Cho-Vega JH, Rassidakis GZ, Amin HM, et al. Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma. Leukemia 2004;18:1872–8.[CrossRef][Medline]
  35. Komyod W, Bauer UM, Heinrich PC, Haan S, Behrmann I. Are STATS arginine-methylated? J Biol Chem 2005;280:21700–5.[Abstract/Free Full Text]
  36. Bode JG, Ludwig S, Freitas CA, et al. The MKK6/p38 mitogen-activated protein kinase pathway is capable of inducing SOCS3 gene expression and inhibits IL-6-induced transcription. Biol Chem 2001;382:1447–53.[CrossRef][Medline]
  37. Canfield S, Lee Y, Schroder A, Rothman P. Cutting edge: IL-4 induces suppressor of cytokine signaling-3 expression in B cells by a mechanism dependent on activation of p38 MAPK. J Immunol 2005;174:2494–8.[Abstract/Free Full Text]
  38. Isobe A, Takeda T, Sakata M, et al. STAT3-mediated constitutive expression of SOCS3 in an undifferentiated rat trophoblast-like cell line. Placenta 2006;27:912–8.[CrossRef][Medline]
  39. Davey GM, Heath WR, Starr R. SOCS1: a potent and multifaceted regulator of cytokines and cell-mediated inflammation. Tissue Antigens 2006;67:1–9.[Medline]
  40. Komazaki T, Nagai H, Emi M, et al. Hypermethylation-associated inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human pancreatic cancers. Jpn J Clin Oncol 2004;34:191–4.[Abstract/Free Full Text]
  41. Fukushima N, Sato N, Sahin F, Su GH, Hruban RH, Goggins M. Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer 2003;89:338–43.[CrossRef][Medline]
  42. To KF, Chan MW, Leung WK, et al. Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line. Br J Cancer 2004;91:1335–41.[CrossRef][Medline]
  43. Nagai H, Naka T, Terada Y, et al. Hypermethylation associated with inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human hepatoblastomas. J Hum Genet 2003;48:65–9.[CrossRef][Medline]
  44. Yoshikawa H, Matsubara K, Qian GS, et al. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 2001;28:29–35.[CrossRef][Medline]
  45. Watanabe D, Ezoe S, Fujimoto M, et al. Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol 2004;126:726–35.[CrossRef][Medline]
  46. Chen CY, Tsay W, Tang JL, et al. SOCS1 methylation in patients with newly diagnosed acute myeloid leukemia. Genes Chromosomes Cancer 2003;37:300–5.[CrossRef][Medline]
  47. Brakensiek K, Langer F, Schlegelberger B, Kreipe H, Lehmann U. Hypermethylation of the suppressor of cytokine signalling-1 (SOCS-1) in myelodysplastic syndrome. Br J Haematol 2005;130:209–17.[CrossRef][Medline]
  48. Galm O, Yoshikawa H, Esteller M, Osieka R, Herman JG. SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood 2003;101:2784–8.[Abstract/Free Full Text]
  49. Herlyn M. Human melanoma: development and progression. Cancer Metastasis Rev 1990;9:101–12.[CrossRef][Medline]
  50. Arcone R, Pucci P, Zappacosta F, et al. Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. Eur J Biochem 1991;198:541–7.[Medline]
  51. Weiergräber O, Hemmann U, Küster A, et al. Soluble human interleukin-6 receptor. Expression in insect cells, purification and characterization. Eur J Biochem 1995;234:661–9.[Medline]
  52. Wegenka UM, Buschmann J, Lütticken C, Heinrich PC, Hom F. Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. Mol Cell Biol 1993;13:276–88.[Abstract/Free Full Text]
  53. Stevenson NJ, Haan S, McClurg AE, et al. The chemoattractants, IL-8 and formyl-methionyl-leucyl-phenylalanine, regulate granulocyte colony-stimulating factor signaling by inducing suppressor of cytokine signaling-1 expression. J Immunol 2004;173:3243–9.[Abstract/Free Full Text]
  54. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
E. C. Brantley, L. B. Nabors, G. Y. Gillespie, Y.-H. Choi, C. A. Palmer, K. Harrison, K. Roarty, and E. N. Benveniste
Loss of Protein Inhibitors of Activated STAT-3 Expression in Glioblastoma Multiforme Tumors: Implications for STAT-3 Activation and Gene Expression
Clin. Cancer Res., August 1, 2008; 14(15): 4694 - 4704.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
E. C. Brantley and E. N. Benveniste
Signal Transducer and Activator of Transcription-3: A Molecular Hub for Signaling Pathways in Gliomas
Mol. Cancer Res., May 1, 2008; 6(5): 675 - 684.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
H. Neuwirt, M. Puhr, I. T Cavarretta, M. Mitterberger, A. Hobisch, and Z. Culig
Suppressor of cytokine signalling-3 is up-regulated by androgen in prostate cancer cell lines and inhibits androgen-mediated proliferation and secretion
Endocr. Relat. Cancer, December 1, 2007; 14(4): 1007 - 1019.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
S. Kreis, G. A. Munz, S. Haan, P. C. Heinrich, and I. Behrmann
Cell Density Dependent Increase of Constitutive Signal Transducers and Activators of Transcription 3 Activity in Melanoma Cells Is Mediated by Janus Kinases
Mol. Cancer Res., December 1, 2007; 5(12): 1331 - 1341.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data, Komyod, et al.
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Komyod, W.
Right arrow Articles by Behrmann, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Komyod, W.
Right arrow Articles by Behrmann, I.

HOME