Lymphoma

Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma

Abstract

Using a cDNA microarray, we found that suppressor of cytokine signaling 3 (SOCS3) is highly expressed in anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) cell lines. As SOCS3 is induced by activated signal transducer and activator of transcription 3 (STAT3), and ALK activates STAT3, we hypothesized that SOCS3 may play a role in ALK+ ALCL pathogenesis via the Janus kinase 3 (JAK3)-STAT3 pathway. Using ALCL cell lines, we show by coimmunoprecipitation experiments that SOCS3 physically binds with JAK3 in vitro, and that JAK3 inhibition by WHI-P154 downregulates SOCS3 expression. Western blot analysis confirmed expression of SOCS3 and also showed coexpression of phosphorylated (activated) STAT3 (pSTAT3). Direct sequencing of the SOCS3 gene showed no mutations or alternative splicing. In ALCL tumors that were assessed by immunohistochemistry, nine of 12 (75%) ALK+ tumors were SOCS3 positive and eight (67%) coexpressed pSTAT3. In comparison, 18 of 25 (72%) ALK-- tumors were SOCS3 positive and seven (28%) coexpressed pSTAT3. These results show that SOCS3 is overexpressed in ALCL, attributable to JAK3-STAT3 activation and likely related to ALK in ALK+ tumors. However, SOCS3 is also expressed in tumors that lack STAT3 and ALK suggesting alternative mechanisms of upregulation.

Introduction

The suppressor of cytokine signaling (SOCS) family of proteins (SOCS1–SOCS7) is characterized by a central src homology 2 (SH2) domain and a C-terminal SOCS box.1 SOCS proteins inhibit many cytokine signaling transduction pathways, including the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway.2 SOCS3 is induced by various cytokines, and its promoter also contains a STAT1/STAT3-binding element.3 These results suggest that SOCS3 is part of a negative feedback loop. Previous studies have shown that SOCS3 has a role as a negative regulator of immune and inflammatory responses in vitro,4, 5 and in particular, is an inhibitor of STAT3 in intestinal inflammation and inflammatory arthritis.6, 7

Anaplastic large cell lymphoma (ALCL), as defined in the World Health Organization (WHO) classification, is a distinct morphologic type of non-Hodgkin lymphoma (NHL) that is heterogeneous at the molecular level.8 A large subset of ALCL is characterized by several chromosomal aberrations that involve the anaplastic lymphoma kinase (alk) gene on chromosome 2p23. These abnormalities result in overexpression of ALK, a tyrosine kinase with known oncogenic potential.9, 10 The t(2;5)(p23;q35), which creates a novel chimeric gene, NPM-ALK, is the most frequently encountered chromosomal abnormality in ALCL. ALK+ ALCL occurs more commonly in young patients who generally have a favorable clinical outcome.8, 11, 12 A second subset of ALCL cases is ALK−. This group is not well defined at the molecular level and others have suggested that ALK− ALCL is better classified as peripheral T-cell lymphoma.11, 13, 14

There is recent evidence that ALK mediates oncogenesis in ALCL via activation of STAT3.15 STAT3 is activated by several mechanisms upon cytokine stimulation16 and activation of STAT3 is mediated by phosphorylation of its tyrosine705 residue. Activated (phosphorylated) STAT3 (pSTAT3) is expressed in most cases of ALK+ ALCL and most likely plays a role in oncogenesis.15, 17 In addition, we have recently shown that JAK3 contributes to STAT3 activation in ALK+ALCL.18 pSTAT3 contributes to the transcription of a wide array of genes involved in controlling cell cycle progression and apoptosis including socs3.7, 19, 20, 21, 22, 23, 24, 25

In a cDNA microarray analysis of 29 lymphoma cell lines, we found that SOCS3 is most highly expressed in ALK+ ALCL cell lines. As SOCS3 is known to be induced by STAT3,4 and ALK is known to activate STAT3,15 we hypothesized that SOCS3, via regulation of the JAK-STAT pathway, may play a role in ALCL pathogenesis. In this study, we show that SOCS3 directly binds to JAK3 in vitro, and that SOCS3 expression is downregulated by inhibition of the JAK-STAT pathway. We also used Western blot and immunohistochemical methods to show that SOCS3 expression is common in ALCL tumors and correlates with STAT3 activation in ALK+ tumors. These results suggest that SOCS3 has a role in ALK+ ALCL lymphomagenesis.

Materials and methods

Lymphoma cell lines

The panel of lymphoma cell lines analyzed by cDNA microarray analysis included three ALK+ ALCL (Karpas 299, SR-786 and SU-DHL-1), four Hodgkin lymphoma (HL) (L428, L1236, MDA-E, and MDA-V), eight diffuse large B-cell lymphoma (DLBCL) (CJ, EJ-1, FL318, JM, JMEA, JP, MS, and SKI-DLCL), six Burkitt lymphoma (BCHN-1, CA46, Daudi, Ramos, Raji, and ST486), four mantle cell lymphoma (JeKo-1, M-1, Mino, and SP53) and four primary (body cavity) effusion lymphoma (BC-1, BC-2, BC-3, and BC-4). All cell lines were grown in suppplemented Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Grand Island, NY, USA) as described previously.17

CDNA microarray

Total RNA was extracted from the cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA). CDNA microarray analysis was performed with a Clontech Human Cancer 1.2 cDNA nylon membrane (Clontech, Palo Alto, CA, USA). Briefly, radiolabeled cDNA probes were synthesized by reverse transcription from 5 μg of total RNA using the manufacturer's protocol with P32-dATP (Amersham Biosciences, Piscataway, NJ, USA). The images were quantified using the Image Quantification Software Array Vision from Imaging Research, Inc. (Ontario, Canada). The signal intensities and local background intensities were determined, and the background-subtracted signal intensities were used for analysis using a program designed at our institution for microarray analysis.26

Western blot analysis

Four ALK+ ALCL (Karpas 299, SR-786, JB-6 and SU-DHL-1), two HL (L-1236 and L-428) and one DLBCL (SKI-DLCL-1) cell lines were examined. Western blot analysis was performed as previously described.17 Primary antibodies specific for SOCS3, pSTAT3tyr705 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (Sigma, St Louis, MO, USA) were used.

Inhibition of JAK3-STAT3 signaling pathway

The ALK+ ALCL cell line, Karpas 299,27 was used in this experiment. In total, 1 × 106 cells/ml were cultured directly with fetal bovine serum (FBS)-free RPMI 1640 medium in tissue culture plates and protected from light. All cell cultures were maintained in an atmosphere of 5% CO2 and 98% humidity at 37°C.

To examine whether inhibition of the JAK3-STAT3 signaling pathway would affect SOCS3 expression, we used a chemical inhibitor of JAK3, WHI-P154 (4-[(3′-Bromo-4′-hydroxyphenyl) amino]-6,7 dimethoxyquinazoline) (Calbiochem, San Diego, CA, USA). WHI-P154 was dissolved in DMSO and kept at −20°C until utilized. The cells were treated for 24 h in the dark with increasing concentrations (10, 20, and 40 μ M) of WHI-P154. Karpas 299 cells treated with DMSO, at a volume equivalent to the volume used for the highest concentration of WHI-P154, were used as a control.

Coimmunoprecipitation studies

Cell lysates from Karpas 299 and SU-DHL1 cell lines were incubated with anti-JAK3 antibody (Santa Cruz Biotechnology), at a concentration of 20 μg/ml overnight at 4°C, and subsequently with protein A/G sepharose (Santa Cruz Biotechnology), for 4 h at 4°C. Four washes with cold PBS and one wash with lysis buffer were then performed, followed by boiling with 25 μl of SDS-PAGE loading buffer (62.5 mM, Tris, pH 6.8; 2% SDS; 5% 2-mercaptoethanol, and 10% glycerol) for 5 min. Thereafter, the samples were stored at −80°C until the time of Western blot analysis. Lysates from both cell lines that were immunoprecipitated with an isotope antibody IgG1 (DakoCytomation) served as negative controls. The experiment was performed twice.

DNA sequencing

Total RNA from Karpas 299 and SU-DHL-1 cell lines was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Further purification of total RNA was carried out with RNeasy columns (Qiagen Inc., Valencia, CA, USA). The first strand cDNA was synthesized from 1 μg of total RNA primed with oligo(dT) following the manufacturer's instructions (Superscript III First-Strand Synthesis System, Invitrogen). The upstream primer was 5′-IndexTermCATGCCCTTTGCGCCCTT and the downstream primer was 5′-IndexTermAGATCCACGCTGGCTCCGT. A 50 μl aliquot of PCR reaction solution was used for the amplification of 5 μl cDNA. One reaction solution contained 1 × PCR buffer, 1.5 mM MgCl2, 0.2 μ M of each primer, 0.2 mM dNTPs, and 2.5 U of Taq polymerase (Invitrogen). The PCR conditions used were 95°C for 30 s, annealing at 63°C for 30 s, and extension at 72°C for 1 min for a total of 45 cycles. Agarose gel electrophoresis and ethidium bromide staining confirmed amplified products. PCR products were purified using the PCR Product Purification Kit (Qiagen, Inc.). The purified DNA fragments were sequenced using ABI PrismTM 377 DNA Sequencer apparatus (PE Applied Biosystems, Foster City, CA, USA).

Immunohistochemistry

Immunohistochemical staining of a tissue microarray of 37 routinely processed ALCL tumors was performed. In total, 12 tumors were ALK+. For comparison, we also assessed five primary cutaneous ALCL, 30 classical HL, and 11 diffuse large B-cell lymphoma. The methods of heat-induced epitope retrieval and the three-step horseradish peroxidase–biotin–streptavidin were described previously.17 The primary antibodies used included SOCS3 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and pSTAT3 (1:25, Santa Cruz Biotechnology). Tumors were considered positive for these proteins when cytoplasmic (SOCS3) or nuclear (pSTAT3) staining was detected in at least 25% of the tumor cells. A cell-block of the Karpas 299 cell line and reactive lymph nodes were used as positive controls.

Results

SOCS3 expression in lymphoma cell lines

CDNA microarray analysis revealed that SOCS3 mRNA levels were highest in ALCL cell lines (Figure 1a). However, two HL and one of six DLBCL cell lines also showed relatively high levels of SOCS3 mRNA (Figure 1a). All three ALK+ ALCL cell lines with high levels of SOCS3 mRNA also had high levels of STAT3 mRNA (Figure 1b).

Figure 1
figure1

SOCS3 and STAT3 expression determined by cDNA microarray in 29 lymphoma cell lines (semilogarithmic scale). (a) SOCS3 mRNA expression was highest in all ALK+ ALCL cell lines, (b) STAT3 was highly expressed in all ALK+ ALCL, one HL, one DLBLC, and one MCL cell lines. SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; ALK, anaplastic lymphoma kinase; ALCL, anaplastic large cell lymphoma; BL, Burkitt lymphoma; PEL, primary effusion lymphoma; HL, Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma; MCL, mantle cell lymphoma.

Using Western blot analysis, we confirmed SOCS3 expression in all ALK+ ALCL cell lines assessed, all of which carry the t(2;5)(p23;q35) (Table 1 and Figure 2). In comparison, three of four HL cell lines showed relatively weaker SOCS3 expression and SOCS3 was not detected in the one DLBCL cell line assessed.

Table 1 Different lymphoma cell lines positive for SOCS3 by cDNA microarray
Figure 2
figure2

Western blot analysis showing coexpression of SOCS3 and pSTAT3 in ALK+ ALCL cell lines. Total cell extracts from four ALK+ ALCL cell lines (Karpas 299, SU-DHL-1, SR-786, and JB-6), four HL cell lines (KMH2, L-1236, L-428, MDA-V), and one DLBCL cell line (SKI-DLCL-1) were transferred and immunoblotted with the indicated antibodies. The arrow indicates the specific band for pSTAT3. β-Actin was used as a control for protein load. SOCS3, suppressor of cytokine signaling 3; pSTAT3, phosphorylated signal transducer and activator of transcription 3; ALK, anaplastic lymphoma kinase; ALCL, anaplastic large cell lymphoma; HL, Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma.

Inhibition of JAK3-STAT3 signaling pathway downregulates SOCS3 in ALCL cells

We analyzed SOCS3 expression in Karpas 299 cells (ALK+ ALCL) after treatment with WHI-P154. WHI-P154 interacts with the Asp-967 residue in the catalytic domain of JAK3 and is likely to bind favorably to JAK3, thereby inhibiting JAK3.18, 28 WHI-P154 treatment resulted in decreased SOCS3 protein levels in a concentration-dependent fashion. SOCS3 was almost entirely absent at a concentration of 40 μ M WHI-P154 (Figure 3). Densitometry studies showed a gradual decrease in the SOCS3 to-β-actin ratio (Figure 3). These findings show that inhibition of the JAK3-STAT3 signaling pathway induces downregulation of SOCS3.

Figure 3
figure3

SOCS3 expression in Karpas 299 cell line after inhibition of JAK3 by WHI-P154. There was substantial, gradual decrease in SOCS3 levels with increasing concentration of WHI-P154. Densitometry studies showed a gradual decrease in SOCS3 to β-actin ratio. SOCS3, suppressor of cytokine signaling 3; JAK3, Janus kinase 3.

SOCS3 directly binds to JAK3 in ALK+ALCL cells

To show if there is any physical binding between SOCS3 and JAK3 proteins, cell extracts of Karpas 299 and SU-DHL-1 cell lines were first immunoprecipitated with anti-JAK3 antibody, and then immunoblotted with anti-SOCS3 antibody. Inversely, lysates from Karpas 299 and SU-DHL-1 cells were also immunoprecipitated with anti-SOCS3 antibody, and then immunoblotted with JAK3 antibody. As shown in Figure 4, SOCS3 was coimmunoprecipitated with anti-JAK3 antibody, indicating that the SOCS3 directly binds with JAK3 in Karpas 299 and SU-DHL1 cells.

Figure 4
figure4

Binding of endogenous SOCS3 with JAK3. Lysates prepared from ALCL cells (Karpas 299 and SU-DHL-1) were first immunoprecipitated with an anti-JAK3 antibody and then analyzed by Western blotting using a monoclonal antibody specific for SOCS3. The SOCS3 panel shows the coimmunoprecipitated SOCS3-JAK3 complex in both ALCL cell lines, compared with lysates of both cell lines that were immunoprecipitated with an isotype IgG1 antibody (negative control). Similar results were obtained when lysates from Karpas 299 and SU-DHL-1 cells were immunoprecipitated with anti-SOCS3 antibody, and then immunoblotted with JAK3 antibody (data not shown). IP, immunoprecipitation; HC, immunoglobulin heavy chain.

Absence of SOCS3 gene alterations or alternative splicing in ALK+ ALCL cells

As SOCS3 expression is thought to be part of a negative feedback loop, we assessed for possible genetic alterations that may contribute to defects in the ability of SOCS3 to downregulate STAT3. We did this by sequencing the SOCS3 gene in Karpas 299 and SU-DHL-1 cell lines. The primers were designed to cover the entire coding region (10246660–10247277, 618 bp) of the SOCS3 gene on chromosome 17q25.3 (GI:37542591). Karpas 299 and SU-DHL-1 cells were sequenced 678 bp (10246644–10247321) and 682 bp (10246640–10247321), respectively. Neither mutation nor alternative splicing of the SOCS3 gene was detected (data not shown).

SOCS3 expression in ALCL tumors

Using immunohistochemical methods, SOCS3 expression was detected in nine of 12 (75%) ALK+ ALCL tumors compared with 18 of 25 (72%) ALK− ALCL tumors (Table 2, Figure 5). SOCS3 immunostaining in all cases was intense with a diffuse cytoplasmic pattern. SOCS3 expression was not found in small reactive lymphocytes. In all six tumors known to carry the t(2;5)(p23;q35), SOCS3 was expressed. By contrast, three tumors had a cytoplasmic pattern of ALK expression, suggesting a variant alk gene abnormality, and only one (33%) case expressed SOCS3 (Table 2).

Table 2 SOCS3 expression in lymphomas
Figure 5
figure5

SOCS3 expression in ALCL and other lymphoma types (a–d). Expression of SOCS3 was localized in the cytoplasm of tumor cells in representative cases of ALK+ ALCL (a, × 400), ALK− ALCL (b, × 400), and DLBCL (c, × 400). In HL (d, × 200), Reed–Sternberg cells (inset, × 400) showed diffuse and strong cytoplasmic expression of SOCS3 protein, but SOCS3 was not expressed in small reactive lymphocytes. SOCS3, suppressor of cytokine signaling 3; ALCL, anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; DLBCL, diffuse large B-cell lymphoma; HL; classical Hodgkin lymphoma.

Coexpression of SOCS3/pSTAT3 in ALCL

All ALK+ ALCL cell lines expressed high levels of SOCS3 and STAT3 at the mRNA (Figure 1) and protein level (Figure 2). In contrast, SOCS3 and STAT3 mRNA were coexpressed in the HL and DLBCL cell lines, but their proteins were not (Figures 1 and 2).

Using immunohistochemistry, eight of 12 ALK+ ALCL tumors coexpressed SOCS3 and pSTAT3 (Table 2), including all six cases with the t(2;5)(p23;q35). All three ALK+ ALCL with cytoplasmic ALK expression and variant genetic lesions did not coexpress SOCS3 and pSTAT3 (Table 2). In ALK− ALCL, coexpression of SOCS3 and pSTAT3 was seen in seven of 25 (28%) tumors (Table 2), with 11 (44%) positive for only SOCS3, four (16%) positive for only pSTAT3, and three (12%) negative for both proteins.

SOCS3 expression in other lymphoma types

SOCS3 expression was detected in three of five (60%) primary cutaneous ALCL, four of 11 (36%) diffuse large B-cell lymphoma, and 23 of 30 (77%) classical HL (Table 2, Figure 5). In positive HL cases, the majority of Hodgkin/Reed–Sternberg cells showed cytoplasmic expression of SOCS3 (Figure 5d inset). Thus, SOCS3 expression is not specific for ALCL. In control reactive lymphoid tissues, SOCS3 was detected in a subset of large centrocytes and centroblasts within reactive germinal centers (data not shown).

Discussion

Cytokine stimulation activates the JAK-STAT pathway, leading to the induction of SOCS proteins. SOCS proteins then inhibit the signaling pathways that initially led to their production as part of a negative feedback loop. SOCS proteins are known to inhibit JAK activity by binding to JAK-proximal sites on cytokine receptors, which subsequently block access of STATs to a receptor-binding site on JAKs.2, 29 STAT3 is a transcription factor with oncogenic potential. There is growing evidence showing that STAT3 may play an important role in the pathogenesis of ALK+ ALCL, and that ALK and JAK3 contribute to STAT3 activation.15, 18, 30 However, the exact mechanisms that control STAT3 activation in these tumors are not entirely understood. In the present study, we focused on SOCS3 in ALCL, because of its role as a major negative feedback regulator of the JAK3-STAT3 signaling pathway. We have shown that ALK+ ALCL cell lines express high levels of SOCS3 mRNA and protein. We have also shown that activated STAT3 is expressed in ALCL and that SOCS3 expression decreases following inhibition of JAK3, suggesting that an active JAK3-STAT3 signaling pathway is required for the expression of SOCS3 in ALCL cells. In addition, we have recently shown that selective inhibition of STAT3 activation using a dominant negative adenoviral vector (adSTAT3DN) results in a decrease of SOCS3 levels in vitro.31 Taken together, the findings suggest that enhanced expression of SOCS3 in ALK+ ALCL is a consequence of preferential activation of STAT3, most likely mediated by ALK and JAK3.

We were particularly interested in the simultaneous strong activation of STAT3 and high levels of SOCS3 mRNA and protein in all ALK+ ALCL cell lines and most ALK+ ALCL tumors. The persistent aberrant activation of STAT3 requires not only defects leading to overproduction but also simultaneous defects in the system normally responsible for turning off activated STAT3. Zang et al32 demonstrated that ALK+ ALCL cells lack protein inhibitor of activated STAT3 (PIAS3) and express protein phosphatase 2A (PP2A), known to maintain STAT3 activation. Thus, in spite of the constitutive expression of SOCS3 in ALK+ ALCL, the persistent activation of STAT3 suggests that SOCS3 expression may not reach a level sufficient to block STAT3 activation, presumably because ALK and JAK3 induce constitutive activation of STAT3. Another possibility is that SOCS3 is functionally defective at the molecular level and therefore it fails to downregulate STAT3 activation. For example, a recent study has reported an N-terminal truncated isoform of SOCS3.33 However, we believe this possibility is unlikely because we did not identify either mutation or alternative splicing of the entire coding region of SOCS3 gene. In addition, we show here for the first time direct binding of SOCS3 to JAK3 in ALK+ ALCL cell lines. Thus, it seems likely that SOCS3 physically interacts with all JAK members, since binding of SOCS3 with JAK1 or JAK2 has been shown previously.2, 29

On the other hand, in 72% of ALK− ALCL, 60% of primary cutaneous ALCL, 77% of HL, and 36% of diffuse large B-cell lymphoma, SOCS3 expression was detected by immunohistochemistry. These neoplasms do not have evidence for involvement of the alk gene in disease pathogenesis.34, 35 In the present study, 44% of ALK− ALCL tumors showed pSTAT3 positivity compared with 92% of ALK+ ALCL. The lack of dependency on ALK for STAT3 activation has been reported in many other human neoplasms that are ALK−, and multiple mechanisms of activating STAT3 also have been identified.19, 36, 37, 38, 39, 40 More specifically, previous studies have shown that STAT3 activation may occur in a subset of ALK− ALCL15, 17 and in a subset of HL.41, 42 In addition to ALK and JAK3, several other oncogenic kinases, such as those encoded by the v-src, abl, v-fps, and v-sis genes, activate STAT3 in a direct and indirect manner.43, 44, 45, 46 This may explain the presence of activated STAT3 in these tumors.

We also show that SOCS3 is overexpressed in lymphomas in the absence of activated STAT3. SOCS3, unlike many of the SOCS proteins, is induced by a broad spectrum of cytokines, including IL-10 and lipopolysacharide.47 Therefore, activation pathways other than JAK-STAT appear to be involved in lymphomas.

In summary, SOCS3 is overexpressed in ALK+ ALCL cell lines and tumors. The correlation of SOCS3 expression with STAT3 activation in ALK+ ALCL, the ability of SOCS3 to bind with JAK3 in vitro, and the dependence of SOCS3 on the JAK-STAT pathway suggest that SOCS3 expression is a result, at least in part, of STAT3 activation, and likely attributable to ALK. However, a subset of cases of ALK− ALCL, HL, and diffuse large B-cell lymphoma also express SOCS3, most likely due to STAT3 activation occurring independently of ALK.

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Correspondence to L Jeffrey Medeiros.

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Cho-Vega, J., Rassidakis, G., Amin, H. et al. Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma. Leukemia 18, 1872–1878 (2004). https://doi.org/10.1038/sj.leu.2403495

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Keywords

  • SOCS3
  • ALCL
  • ALK
  • STAT3
  • JAK3

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