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NEOPLASIA
From the Institute of Medical Microbiology and
Immunology, University of Copenhagen, Copenhagen, Denmark; Institute of
Human Genetics, University of Aarhus, Aarhus, Denmark; Department of
Pathology and Laboratory Medicine, University of Pennsylvania Medical
Center, Philadelphia, PA; and Signal Transduction, Novo Nordisk
Discovery, Bagsværd, Denmark.
A characteristic feature of neoplastic transformation is the loss
of external control by cytokines and extracellular matrix of cellular
differentiation, migration, and mitogenesis. Because suppressors of
cytokine signaling (SOCS) proteins are negative regulators of
cytokine-induced signaling, it has been hypothesized that an aberrant
SOCS expression plays a role in neoplastic transformation. This study
reports on a constitutive SOCS-3 expression in cutaneous T-cell
lymphoma (CTCL) cell lines. SOCS-3 protein is constitutively expressed
in tumor cell lines (but not in nonmalignant T cells) obtained from
affected skin from a patient with mycosis fungoides (MF) and from
peripheral blood from a patient with Sezary syndrome (SS). In contrast,
constitutive SOCS-3 expression is not found in the leukemic Jurkat
T-cell line, the MOLT-4 acute lymphoblastic leukemia cell line, and the
monocytic leukemic cell line U937. Expression of SOCS-3 coincides with
a constitutive activation of STAT3 in CTCL tumor cells, and stable
transfection of CTCL tumor cells with a dominant negative STAT3
strongly inhibits SOCS-3 expression, whereas transfection with
wild-type STAT3 does not. Moreover, the reduced SOCS-3 expression in
cells transfected with the dominant negative STAT3 is associated with
an increased sensitivity to interferon- A characteristic feature of neoplastic
transformation is the loss of external control of cellular
differentiation, migration, and mitogenesis. Via an autocrine
production of growth factors or a constitutive activation of
intracellular signaling pathways, tumor cells can gain independence of
external growth factors. Moreover, through the development of
resistance to cytokines, transformed cells can evade the influence of
regulatory growth and differentiation signals.1 In early
stage melanoma cell lines interleukin (IL)-6 functions as a growth
inhibitor, but during tumor progression melanoma cells develop
resistance to IL-6, and advanced-stage melanoma cell lines are
resistant to growth inhibition by IL-6.2,3 Likewise, many
tumors develop resistance to interferon- The suppressors of cytokine signaling (SOCS) proteins comprise a
recently identified family of negative regulators of cytokine signaling
(also referred to as the CIS/JAB or SSI family).6-8 They
are characterized by a central SH2 domain and a C-terminal SOCS-box,
and the SOCS family currently consists of 8 members (CIS and SOCS-1 to
SOCS-7).9 Among the 8 family members, SOCS-1 and SOCS-3
are the most potent inhibitors of cytokine-induced signaling.
Transfection experiments with overexpression of the relevant SOCS have
demonstrated that SOCS-1 and to a lesser extent SOCS-3 are able to
suppress signaling induced by an array of cytokines, including among
others IL-2, IL-4, IL-6, IFN- Normally, transcription of SOCS genes is induced after
stimulation with cytokines, and the expression of SOCSs is generally short-lived.21 The finding of STAT binding sites in the
CIS, SOCS-1, and SOCS-3 promoters8,18,24 as well as the
discovery that transfection with dominant negative STAT3 inhibits
cytokine-induced expression of SOCS-1 and
SOCS-38,24 indicates that STAT transcription factors play
an essential role in transcription of the SOCS genes. Aberrant STAT activation is found in many malignancies,25
and recently STAT3 was identified as being an oncogene
itself.26 However, only little is known about the
expression and possible function of SOCS proteins in tumor cells. In
this study, we addressed whether abnormal STAT3 activation mediates
SOCS expression in tumor cells. We show that tumor cell lines
established from patients with cutaneous T-cell lymphoma (CTCL) have a
constitutive expression of SOCS-3. SOCS-3 expression in these cells
correlates with a constitutive activation of STAT3 and by transfection
with a dominant negative STAT3, we show that SOCS-3 expression depends
on a fully functional STAT3. Moreover, we show that the decrease in
SOCS-3, caused by the dominant negative STAT3, correlates with an
increased IFN- Antibodies and other reagents
Cell lines and culture
Protein extraction and Western blotting For protein extraction, cells were rapidly pelleted and lysed in ice-cold lysis buffer as described previously.32 The Western blotting procedure is described in detail elsewhere.33 Blots were evaluated using enhanced chemiluminescence according to the manufacturer's manual (Amersham, Buckinghamshire, UK).Analysis of messenger RNA (mRNA) levels Total cellular RNA was isolated using the RNeasy plant mini kit (Qiagen, Hilden, Germany). The RNA samples were treated with DNase (Promega, Madison, WI), followed by reverse transcription (RT) with Superscript II (Life Technologies) performed with 1 µg RNA, 3 µg Random primer (Life Technologies), 0.25 mM dNTP (Amersham), 10 U RNasine (Promega), and DTT and reaction buffer supplied with the reverse transcriptase. RT-negative controls were run in every polymerase chain reaction (PCR) experiment and did not yield a product, indicating the absence of contaminating genomic DNA. A total of 2% of each cDNA was subjected to hot start PCR performed with DyNAzyme II DNA polymerase (Finnzymes, Espoo, Finland) for amplification of CIS, SOCS-2, and SOCS-3, or with HotStarTaq DNA polymerase (Qiagen) for amplification of SOCS-1. Hot start PCR with the DyNAzyme II DNA polymerase was carried out by initial separation of the DNA polymerase from the rest of the reaction mixture, using DyNA wax (Finnzymes). Each PCR reaction was carried out with 0.2 µM of the relevant primers, 40 µM dATP, dGTP, and dTTP, 20 µM dCTP, 1.25 µ Ci [ -33P]dCTP (Amersham), and reaction buffers supplied
with the respective DNA polymerases. Preliminary PCR experiments showed
that the rate of amplification was linear for CIS, SOCS-1, SOCS-2,
SOCS-3, and TBP (TATA binding protein = transcription factor
IID) when applying fewer than 28 cycles (data not shown), and
we chose to use 24 cycles in PCR experiments with CIS, SOCS-2, and
SOCS-3 primers, and 26 cycles in PCR experiments with SOCS-1 primers.
The PCR experiments with DyNAzyme consisted of an initial denaturation step (95°C, 10 minutes), followed by 24 cycles (denaturation 95°C, 30 seconds; annealing 55°C, 1 minute; elongation 72°C, 1 minute) and a single elongation step at 72°C for 7 minutes, whereas the PCR
experiments with HotStarTaq were carried out with an initial denaturation step (95°C, 15 minutes), followed by 26 cycles
(denaturation 94°C, 1 minute; annealing 55°C, 1 minute; elongation
72°C, 1 minute) and a single elongation step at 72°C for 10 minutes. The reaction mixtures were then mixed with loading buffer
(0.03% bromophenol blue and 0.03% xylene cyanole in formamide) and
loaded onto a 5% urea-acrylamide sequencing gel. The gels were
subsequently exposed to a Kodak biomax film (Amersham) or to
33P quantification by Phosphorimager analysis. DNA marker V
(Boehringer Mannheim, Mannheim, Germany) and DNA marker VI (Boehringer
Mannheim) were labeled using a 5'-end labeling kit (Amersham) and
[ -33P]ATP (Amersham) according to the manufacturer's
manual. TBP was used as an internal standard to correct for
unequal pipeting or loading or both, and SOCS/TBP is used as the
relative SOCS expression. The following primers were used for the
specific amplification of CIS, SOCS-1, SOCS-2, SOCS-3, and TBP:
CIS 5'-cggctggactctaactgct-3' and 5'-aaggggtactgtcggaggta-3'
(350-bp product), SOCS-1 5'-cacgcacttccgcacattc-3' and
5'-agcagctcgaggaggcagtc-3' (297-bp product), SOCS-2
5'-caggatggtactggggaagt-3' and 5'-tggacctgtccgcttatc-3' (284-bp
product), SOCS-3 5'-gagccccctccttcccctcgc-3' and
5'-ggtccaggaactcccgaatg-3' (264-bp product), TBP
5'-tgacccccatcactcctg-3' and 5'-ggttcgtggctctcttatcc-3'
(191-bp product).
Constitutive expression of SOCS mRNA in CTCL cell lines To investigate the expression of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA in CTCL tumor cell lines, total cellular RNA was subjected to RT-PCR and quantitated as described in "Materials and methods." As shown in Figure 1, a high constitutive expression of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA was found in 2 tumor cell lines (MyLa 2000 and MyLa 3675) obtained from affected skin from a patient with MF, which is the most common form of CTCL. The late culture MF tumor cell line (MyLa 2000) expressed higher levels of SOCS-3 and lower levels of CIS and SOCS-2 as compared to the early culture cell line (MyLa 3675), whereas the relative SOCS-1 expression was comparable between the 2 cell lines (Figure 1B). Jurkat cells, which is a leukemic T-cell line, were included in the experiments to address whether a constitutive expression of SOCS mRNA is found in all malignant T-cell lines. Of the 4 SOCSs of interest, only SOCS-2 was readily detected in Jurkat cells (Figure 1A, lane 12). However, this expression of SOCS-2 is low compared to the ones found in MyLa 2000 and MyLa 3675. Essentially identical findings were obtained in 3 additional, independent experiments (Figure 1C).
Constitutive expression of SOCS-3 protein in CTCL tumor cell lines but not in nonmalignant cells To address whether the constitutive SOCS expression is also evident at the protein level in the CTCL tumor cell lines, whole cell lysates from MyLa 3675 and MyLa 2000 as well as from an autologous nonmalignant T-cell line (MyLa 1885) were analyzed by Western blotting with SOCS-specific antibodies. As shown in Figure 2A, SOCS-3 protein, which appears as a double band, was expressed in both tumor cell lines but not in the nonmalignant cell line. Moreover, in agreement with the differences in SOCS-3 expression, found at the mRNA level, expression of SOCS-3 protein was higher in MyLa 2000 as compared to MyLa 3675. Stripping and reprobing of the membrane with antibodies directed against the ERK1/2 and p38 MAP kinases showed that the malignant and nonmalignant cell lines express comparable amounts of these MAP kinases, suggesting that the differences in SOCS-3 expression were not caused by differences in the amount of loaded protein.
Because the MF tumor cell lines have a constitutive active STAT3,33 and because STAT3 is implicated in the cytokine-induced expression of SOCS-3,24 the cell lysates were also analyzed using an antibody directed against tyrosine phosphorylated (active) STAT3 (PY-STAT3). In accordance with previous findings,33 the late culture cell line (MyLa 2000) has a higher constitutive level of STAT3 phosphorylation than the early culture cell line (MyLa 3675), even though the 2 cell lines express equal amounts of STAT3 (Figure 2B). The differences in STAT3 activation correlate with the differences in SOCS-3 expression, and the constitutive SOCS-3 expression could thus be a consequence of the constitutive active STAT3. Although CIS, SOCS-1, and SOCS-2 mRNA were detected in both MF tumor cell lines, we were unable to detect these SOCSs at the protein level when using antibodies, which were able to detect SOCS expression in 293 cells transiently transfected with the particular SOCS (data not shown). Because serum preparations might contain cytokines/growth factors, STAT3 activation and SOCS-3 expression were also examined in serum-deprived cells. However, neither STAT3 activation nor SOCS-3 expression was decreased in these cells (data not shown), thereby confirming that the observed activation/expression is not a consequence of a serum-contained factor. Constitutive SOCS-3 expression in CTCL cells from an SS patient but not in other hematopoietic tumor cell lines Next, we looked for SOCS-3 expression in a cell line (SeAx 4405) established from a patient with SS, which is a leukemic variant of CTCL. Like the MF tumor cell lines, the SeAx 4405 cell line also has a constitutive active STAT3 (K. Eriksen, manuscript submitted). Total cellular RNA from SeAx 4405 cells as well as from the nonmalignant T-cell line, MyLa 1885, and a bulk culture of human thymocytes, were subjected to RT-PCR and quantitated as above. As shown in Figure 3, SeAx cells have a high constitutive expression of SOCS-3 mRNA. In contrast, the thymocytes and the nonmalignant MyLa 1885 cells express little SOCS-3 mRNA, the latter being in accordance with the lack of SOCS-3 protein expression in MyLa 1885, depicted in Figure 2A.
To address whether SOCS-3 is also evident at the protein level in the
SeAx cells, Western blotting experiments were performed with the
anti-SOCS-3 antibody as above. As shown in Figure 4, SOCS-3 protein is
constitutively expressed in SeAx 4405 cells (lane 1), whereas it is not
detected in other hematopoietic tumor cell lines including the Jurkat
cells, an acute lymphoblastic leukemia cell line (MOLT-4), and a
monocytic leukemia cell line (U937) (lanes 2-5). The membranes were
also examined using the antibody directed against the active STAT3. It
was hereby confirmed that the SeAx cells have a constitutive active
STAT3. Furthermore, it was shown that the tumor cell lines, which have
no expression of SOCS-3, also do not have an active STAT3. The amount
of total STAT3 varied among the cell lines (Figure 4, middle row), but by reprobing with antibodies against the ERK1/2 and p38 MAP kinases, the loading of equal amounts of lysates was confirmed.
Activated STAT3 mediates SOCS-3 expression in CTCL cells The results shown in Figures 2 and 4 indicated that there is a correlation between STAT3 activation and SOCS-3 expression in CTCL tumor cells, which is in agreement with the finding that STAT3 is implicated in the LIF-induced SOCS-3 expression in AtT-20 cells.24 As shown in Figure 5, inhibition of the tyrosine phosphorylation of STAT3 by a JAK3 inhibitor is associated with an inhibition of SOCS-3 expression. Indeed, the constitutive SOCS-3 expression is almost completely blocked at concentrations of 100 µg/mL, that is, at concentrations that block the constitutive tyrosine phosphorylation of STAT3. To address more directly the involvement of STAT3 in SOCS expression in CTCL tumor cells, SOCS expression was investigated in MyLa 2000 cells stably transfected with either a dominant negative STAT3 (ST3D) or wild-type STAT3 (ST3WT). SOCS mRNA expression was compared in 6 independent ST3D and ST3WT transfectant cell lines. As shown in Figure 6A-D, ST3D and ST3WT cells express comparable levels of CIS, SOCS-1, and SOCS-2 mRNA, whereas ST3D cells express lower levels of SOCS-3 mRNA as compared to ST3WT cells. Essentially identical results were obtained in an additional, independent experiment, and as shown in Figure 6H, the expression of SOCS-3 mRNA is decreased in all 3 ST3D cell lines as compared to the ST3WT cell lines.
To investigate whether the effect of the dominant negative STAT3 on
SOCS-3 expression is also evident at the protein level, whole cell
lysates from the ST3D and ST3WT cell lines plus from untransfected MyLa
2000 cells, were analyzed. In agreement with the results obtained at
the mRNA level, SOCS-3 protein expression is clearly affected in the
cells expressing the defective STAT3 (Figure
7). In contrast, the expression of p38
MAP kinase was comparable in all transfectant cell lines and in
untransfected cells.
Correlation between SOCS-3 expression and IFN- and also
to IFN-, although to a lesser extent.14 If the
constitutive SOCS-3 expression in CTCL tumor cells affects the IFN
sensitivity of the cells, then ST3D cell lines would be more sensitive
to IFNs as compared to ST3WT cell lines, due to the large difference in
SOCS-3 expression. To investigate this, whole cell lysates from cells
stimulated with either IFN- or IFN- were analyzed by Western
blotting with an antibody, which recognizes the tyrosine phosphorylated
(active) STAT1 (PY-STAT1). Both IFN- and IFN- induce activation
of STAT1, and as can be seen from Figure
8A, IFN- induces a higher activation of STAT1 in ST3D14 cells than in ST3WT6.2 cells (upper row, compare lane 3 and 6). In contrast, the STAT1 activation in response to IFN-
is similar in the 2 cell lines (lanes 2 and 5). Stripping and reprobing
the membrane with antibodies against STAT1, p38 MAP kinase, and SOCS-3
confirmed the loading of equal amounts of lysate as well as the
difference in SOCS-3 expression between the 2 cell lines (the very weak
appearance of the upper STAT1 band in ST3D14 was not seen in other
experiments). Besides activation of STAT1, IFN- also induces STAT3
activation in certain cells.34 Analysis of the STAT3
activation in ST3D14 and ST3WT6.2 cells showed that IFN- induces a
markedly higher level of STAT3 activation in ST3D14 cells than in
ST3WT6.2 cells (Figure 8B). Due to a higher expression of the exogenous
STAT3, the ST3D14 cells express higher amounts of STAT3 as compared to
ST3WT6.2 cells. However, this difference in STAT3 expression does not
account for the large difference in IFN--induced STAT3 activation.
Thus, the increased IFN- sensitivity in ST3D14 cells is evident when
looking at both STAT1 and STAT3 activation levels. To ascertain that
the increased IFN- responsiveness in the ST3D14 cells was not due to
alterations in receptor expression, IFN- receptor expression was
analyzed. No differences in receptor expression were found between the
ST3D14 and ST3WT6.2 cell lines (data not shown).
In the present study, we show that CTCL lines constitutively express SOCS-3. Thus, SOCS-3 expression was detected in 3 CTCL tumor cell lines established from patients with MF and SS, whereas it was absent in autologous nonmalignant T cells as well as in tumor cell lines from other malignancies. Because SOCS-3 expression seemed to correlate with STAT3 activation, we addressed whether the constitutive SOCS-3 expression was mediated by STAT3. Indeed, inhibition of STAT3 tyrosine phosphorylation by an inhibitor of JAK kinases correlated with inhibition of SOCS-3 expression. More importantly, stable transfection of CTCL cells with a dominant negative STAT3 provided direct evidence for the implication of STAT3 in the constitutive expression of SOCS-3. This conclusion is in agreement with the recent findings that STAT3 regulates cytokine-induced SOCS-3 transcription24 as well as the present observation that SOCS-3 was not expressed in malignant and nonmalignant cell lines, which did not have a constitutive active STAT3. In a recent paper, Schuringa and colleagues35 reported on constitutive expression of SOCS-1 and SOCS-3 mRNA in acute myelogenous leukemia (AML) cells, and in agreement with our observations, they were only able to detect SOCS expression in cells with a constitutive active STAT3. However, they were unable to show a direct association between STAT3 activation and SOCS expression. Moreover, they did not address whether SOCS mRNA was translated into protein and played a functional role in AML cells. The CTCL tumor cell lines also had a high constitutive expression of CIS, SOCS-1, and SOCS-2 mRNA. We were, however, unable to detect these SOCSs at the protein level in these cell lines. Thus, CIS, SOCS-1, and SOCS-2 proteins were undetectable in Western blotting using antibodies recognizing the SOCS of interest. Although we cannot formally rule out the possibility that CIS, SOCS-1, and SOCS-2 proteins are expressed at low levels in CTCL tumor cell lines, our observations suggest that these SOCS mRNAs may not be efficiently translated into protein. It was recently shown that SOCS-1 expression, in addition to regulation at the transcriptional level, is also regulated at the translational level.36 Alternatively, posttranslational modifications might mask epitopes recognized by the CIS-, SOCS-1-, and SOCS-2-specific antibodies. Because SOCS-3 appeared as a double band in Western blotting, it is possible that CTCL cells express 2 SOCS-3 isoforms or that SOCS-3 is subjected to posttranslational modifications such as proteolytic cleavage or phosphorylation in these cell lines. SOCS-3 becomes tyrosine phosphorylated in response to IL-2 and EPO stimulation,10,23 and it was shown that JAK1, JAK2, and Lck are able to phosphorylate SOCS-3.10 We have, however, been unable to detect tyrosine phosphorylation of SOCS-3 in the CTCL tumor cells, suggesting that posttranslational modifications other than tyrosine phosphorylation are responsible for the appearance as a double band. Recently, STAT3 was shown to function as an oncogene in
vivo26 and a constitutive activation of STAT3 has been
reported in a number of hematologic and nonhematologic
malignancies.25 Although STAT3 has been implicated in the
regulation of several genes involved in cell cycle regulation, cell
differentiation, and apoptosis,37 it is not fully
understood how an aberrant STAT3 activation drives cells to undergo
malignant transformation. The present finding that STAT3 mediates a
constitutive expression of SOCS-3 in CTCL tumor cell lines, suggests
the possibility that SOCS-3 plays a role in STAT3 mediated oncogenesis.
A characteristic feature of malignant transformation is the loss of
external control from cytokines or other factors,1 and
experimental data show that tumors can evade the influence of
regulatory growth and differentiation signals through development of
resistance to cytokines.2,3 It is possible that a
constitutive SOCS-3 expression changes cytokine receptor sensitivity or
the balance between different cytokine responses in a way that promotes
malignant transformation. In this report, we show that the decreased
SOCS-3 expression in CTCL cells expressing a dominant negative STAT3 is
associated with an increased IFN- In conclusion, we provide evidence of a high constitutive SOCS
expression in cancer cell lines established from CTCL patients. Our
results show that the aberrant SOCS-3 expression is mediated by STAT3,
suggesting that this might be a general feature for cancer cells with a
constitutive active STAT3. Furthermore, our results indicate that the
aberrant SOCS-3 expression affects the IFN-
We thank Koichi Nakajima for providing the vectors containing wild-type and dominant negative STAT3.
Submitted July 6, 2000; accepted October 11, 2000.
Supported in part by grants from the Weimann Foundation (M.N.), the Benzon Foundation, the Carlsberg Foundation, the Leo Dannin and wife legat, the Danish Medical Research Council, the Novo Nordic Foundation, the Danish Medical Associations Research Foundation, the Danish Cancer Research Foundation, the Danish Cancer Society, and the National Institute of Health (grant no. CA89194). C.B. was a recipient of a scholarship from the Danish Cancer Society.
C.B. and M.N. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Niels Ødum, Institute of Medical Microbiology and Immunology, Panum 22.5, University of Copenhagen, Blegdamsvej 3, DK2200 Copenhagen N, Denmark; e-mail: n.odum{at}immi.ku.dk.
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© 2001 by The American Society of Hematology.
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Abstract
Introduction
