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Mutations of the tumor suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation

Abstract

The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P. For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here, we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (P<0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.

Main

There is increasing evidence that loss of function of suppressor of cytokine signaling (SOCS)-1 is critically involved in the development and progression of several specific cancers (Rottapel et al., 2002; Sutherland et al., 2004). In various tumors, for example, multiple myeloma (Galm et al., 2003; Chim et al., 2004a), acute myeloid leukemia (Chen et al., 2003), mantle cell, and follicular lymphoma (Chim et al., 2004b), and in hepatocellular, pancreatic, ovarian, and breast carcinomas (Yoshikawa et al., 2001; Komazaki et al., 2004; Sutherland et al., 2004) the SOCS-1 gene was shown to be silenced by aberrant DNA methylation. In addition to the frequent epigenetic imprinting of tumor suppressor genes, we recently detected as a novel finding deletion mutations of SOCS-1 in primary mediastinal B-cell lymphoma (PMBL) predominantly leading to a truncation of the predicted SOCS-1 proteins (Melzner et al., 2005). Furthermore, we showed that the PMBL line MedB-1 has a biallelic mutation leading to abrogation of the SOCS box domain, and that PMBL line Karpas1106P has a biallelic deletion within chromosomal region 16p13.13 encompassing the SOCS-1 locus, hence, is SOCS-1−/− (Melzner et al., 2006).

The SOCS protein family consists of eight members that share a central Src-homology 2 (SH2) domain and a highly conserved C-terminal domain termed SOCS box (Endo et al., 1997; Hilton et al., 1998). SOCS-1 downregulates the kinase activity of Janus kinases (JAK1, JAK2, JAK3, and TYK2) mediated by binding via its SH2 domain to the catalytic center of the phosphorylated JAKs. The SOCS box of SOCS-1 associates with the elongins B and C to the elongin BC complex, whereupon SOCS proteins and their substrates are subjected to proteasomal degradation. In the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway the downstream STAT molecules reside in the cytoplasm until activated via phosphorylation of activated JAKs. After dimerization of the phospho-STATs, they finally enter the nucleus and modulate the transcription of several target genes. This pathway is induced by a variety of cytokines and growth factors and involved in multiple cellular processes, for example, proliferation, development, differentiation, and survival. Since SOCS-1 is a target gene of STATs its generally low expression is rapidly upregulated upon cytokine-induced JAK/STAT signaling. Therefore, SOCS-1 exerts the function of a negative feedback regulator of STAT action (Krebs and Hilton, 2001).

Constitutive activation of STAT3, STAT5, and STAT6 strongly indicates altered JAK/STAT signaling and has been reported for several lymphomas including PMBL and classical Hodgkin lymphoma (cHL) (Kube et al., 2001; Skinnider et al., 2002; Guiter et al., 2004). PMBL is a rare disease with characteristic features distinguishing it from other types of diffuse large B-cell lymphoma (Barth et al., 2002), however, there is increasing evidence of a relatedness to the far more frequent cHL. One discriminative criteria and at the same time major obstacle for studying cHL is the low abundance of the neoplastic Hodgkin and Reed-Sternberg (HRS) cells within the tumor tissue that mainly consists of reactive nonmalignant cells. Despite clear differences in age distribution, sex predominance, lymphoma spread, histology, and immunophenotype of the neoplastic cells between PMBL and cHL, there is a growing list of common characteristic hallmarks like mediastinal (thymic) origin, loss of MHC class I presentation, lack of immunoglobulin expression despite successfully rearranged IgH genes, lack and/or functional Oct2 deficiency, and frequent gains of 2p13-p16 and 9p24 involving REL and JAK2, respectively (Joos et al., 1996, 2003; Barth et al., 2003; Savage et al., 2003; Ritz et al., 2005). Furthermore, two extensive studies on gene expression profiling revealed substantial similarities between PMBL and cHL cell lines in terms of commonly up- or downregulated genes (Rosenwald et al., 2003; Savage et al., 2003).

As a functional readout of our previous study, we showed that the mutations of SOCS-1 in the PMBL line MedB-1 led to a delayed degradation of the JAK2 protein and a sustained action of phospho-JAK2 and ultimately to constitutive activation of JAK/STAT signaling. Moreover, ectopic expression of wild-type (wt) SOCS-1 in MedB-1 cells dramatically decreased the levels of phosphorylated JAK2 and STAT5, and significantly reduced the proliferation rate of MedB-1 cells (Melzner et al., 2005). Taking the above listed similarities between PMBL and cHL into account, it was obvious to investigate SOCS-1 status in cHL.

To this end, we laser-microdissected 20 neoplastic Hodgkin and Reed-Sternberg (HRS) cells from tumor tissue of 19 cHL and performed DNA isolation. We also took advantage of five HL-derived cell lines (HDLM-2, L1236, L428, KM-H2, and L540), isolated DNA and RNA and subjected those to sequencing analysis.

DNA sequencing analysis of the cell line HDLM-2 revealed a deletion mutation in one SOCS-1 allele while the second allele did not exhibited a mutation. Intriguingly, transcriptional data displayed an exclusive expression of the mutated allele (Figure 1a). Thus, the expression of the wt SOCS-1 allele was apparently silenced. Of note, Western Blot analysis of whole cell lysates of HDLM-2 cells showed high levels of phospho-STATs (Skinnider et al., 2002). Considering our data, this might be due to the mutation and loss of function of one SOCS-1 allele and lack of expression of the second allele. Like in the PMBL line MedB-1, we detected biallelic SOCS-1 mutations in the HL lines L1236 and L428 and also found the corresponding transcripts by RT–PCR (Figure 1). Due to an in-frame mutation of one SOCS-1 allele in L428 and in L1236, the corresponding protein sequences lack five and four internal amino acid (aa) residues, respectively, so that the deletions are in close proximity within the SH2 domain leaving the SOCS box sequence unaffected. In contrast to MedB-1, however, recent data showed that L428 cells have no delayed JAK2 degradation arguing for the translation of the mutated SOCS-1 transcript in L428 into an at least partly functional protein (Melzner et al., 2005).

Figure 1
figure1

Mutational analysis of SOCS-1 in HL-derived cell lines. L428, KM-H2, HDLM-2, and L540 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. L1236 was kindly provided by V Diehl, University of Cologne, Cologne, Germany. All cell lines were grown in RPMI 1640 medium with 10% fetal calf serum (FCS) supplemented with penicillin/streptomycin (1%) and glutamine (1%) and were maintained at 37°C in a 5% CO2 containing atmosphere. (a) Gel view of a DNA 500 LabChip™ utilizing the Agilent Bioanalyzer (Agilent, Waldbronn, Germany). PCR was performed on (a) DNA and (b) cDNA prepared from the indicated HL cell lines using a primer pair amplifying a 369 bp fragment (nt 167–535) of the wt SOCS-1 coding sequence. DNA was extracted from the obtained bands and subjected to sequencing analysis. (b) Detailed sequencing chromatogram of the mutated SOCS-1 cDNA sequencs in HDLM-2, L1236, and L428 cells. The wt SOCS-1 sequences are superposed and the deleted regions are marked. DNA sequences are numbered in relation to the start codon as 1, and the corresponding protein sequences are shown.

We found mutations of SOCS-1 in three of five HL cell lines and detected SOCS-1 mutations in 8/19 cHL samples analysed. The laser-microdissected HRS cells of eight cases exhibited SOCS-1 mutations as shown by DNA sequencing analysis. In Figure 2 the effects of the SOCS-1 mutations on the protein sequences are illustrated. In the majority of the HRS cells of these cHL's, we detected out-of-frame deletions that cause premature stop codons. The predicted aa sequences, therefore, yielded shortened SOCS-1 proteins lacking parts of or the complete C-terminal SOCS box domain.

Figure 2
figure2

SOCS-1 protein structure and SOCS-1 mutations. Tumor tissue was drawn from our bank of fresh frozen tissue. The tumor material was pseudonymized to comply with the German law for correct usage of archival tissue for clinical research (Deutsches Ärzteblatt 2003; 100 A1632). Approval for this procedure was obtained from the local ethics committee. To selectively isolate tumor cell DNA, a micro-methodology was applied. HRS cells from tumor tissues were laser-microdissected using the PALM MicroBeam system (PALM Microlaser Technologies, Bernried, Germany). Likewise, we isolated 20 neoplastic cells per HL cell line from cytospin preparations. These isolated cells were catapulted into the mineral-oil coated cap; 20 μl of 1 × PCR buffer (Qiagen) containing 200 μg/ml proteinase K were added and incubated overnight at 42°C. DNA amplification was performed as previously described (Melzner et al., 2005). For RNA analysis we refered to HL cell lines only. Reverse transcription PCR and sequencing analyses of SOCS-1 and of SOCS-1 cDNA were performed as previously described (Melzner et al., 2005). (a) the N-terminal domain of SOCS-1 contains a proline rich region (P), a kinase inhibitory region (KIR) spanning from aa 55 to aa 66 and an extended SH2 subdomain (ESS) from aa 67 to aa 78. KIR, ESS and SH2 domains are required for JAK binding and inhibition. The C-terminal region from aa 161 to aa 210 is termed SOCS box and contains a 10 aa consensus sequence for interaction with the elongin BC complex. (b) predicted SOCS-1 protein sequences of SOCS-1 in HL-derived cell lines. The coding sequences of SOCS-1 were amplified by PCR using DNA from HDLM-2, L1236, and L428 cells as template and subjected to sequencing analysis. The protein sequences related from the DNA sequences are schematically shown. The positions of mutations in SOCS-1 DNA and its consequences on protein sequence are indicated (del , deletion; fs, frame shift; nonsense sequence, - - - - -). (c) SOCS-1 mutations found in cHL. The SOCS-1 protein sequences of the cHL cases related from their DNA sequences are presented. The wt SOCS-1 protein sequence is superposed and the essential domains are boxed and shaded. Nonsense sequence, - - - - -; premature STOP codon is marked by an asterisk.

Comparable to the predicted SOCS-1 protein of cell line L428, cHL case no. 1 has an in-frame deletion of 15 nucleotides, thus, the predicted protein sequence lacks five internal aa residues not impairing the SOCS box domain. In cHL cases no. 2–8 the SOCS-1 mutations affected parts of the SH2 domain and led to a complete loss of the SOCS box sequence. Similar loss of function mutations, as shown in PMBL cell line MedB-1, resulted in high cellular levels of phospho-STAT5. These were dramatically reduced upon ectopic expression of wt SOCS-1 in these cells. In order to determine the state of phospho-STAT5 in the cHL samples we performed immunhistochemistry on paraffin sections. In the HRS cells of all cHL samples with SOCS-1 mutation we found phospho-STAT5 located in the cytoplasm and in six of eight cases phospho-STAT5 accumulation in the nucleus (Figure 3). This nuclear accumulation of phosphorylated STAT5 was positively associated with SOCS-1 mutations (P<0.01, Fisher's exact test) and per se indicates constitutive activation of phospho-STAT5 in the HRS cells of cHL tissue. Since SOCS-1 was found to be hypermethylated and thereby silenced in several types of lymphomas (Chen et al., 2003; Galm et al., 2003; Chim et al., 2004a, 2004b), it is tempting to speculate that the two cHL cases without mutations of the SOCS-1 gene but detectable phospho-STAT5 might have downregulated SOCS-1 expression due to aberrant DNA methylation or other mutations that constitutively activate STAT5.

Figure 3
figure3

Immunohistochemical analysis of phospho-STAT5 in HRS cells of cHL. Immunohistochemistry (IHC) was performed on cryostat sections or on formalin-fixed, paraffin-embedded tissue sections as previously described (Barth et al., 2003). Antibody against phospho-STAT5 (Cell Signaling Technology, Beverly, MA) was applied to IHC in a dilution of 1:50. IHC results were analysed for association with the mutational status using Fisher's exact test. (A) tabular list of the phospho-STAT5 IHC results including cHL subtypes of nodular sclerosis (NS), mixed cellularity (MC), lymphocyte-rich (LR), or lymphocyte-depleted (LD) and their SOCS-1 status: +, mutated or −, not mutated. IHC evaluation: −, negative staining; +, staining in up to 30%; ++, staining in more than 30% and up to 70%; +++, staining in more than 70% of HRS cells; ND, not done; NE, not evaluable. (B) phospho-STAT5 IHC on paraffin-embedded tumor tissue of two cHL cases. (a), phospho-STAT5 in HRS cells of cHL case no. 5 is cleary located in the nucleus and in the cytoplasm as highlighted in the insert. (b), in cHL case no. 9 with wt SOCS-1 no phospho-STAT5 stained HRS cells are found (bar=50 μm).

Recently, constitutive kinase activity due to the gain of function mutation V617F in JAK2 has been shown to increase phospho-STAT5 level by ectopic expression of the V617F JAK2 variant in BaF/3 cells and was frequently found in myeloproliferative disorders (James et al., 2005; Kralovics et al., 2005). Therefore, we checked DNA of the HL lines and the PMBL lines MedB-1 and Karpas1106 by site-specific restriction analysis for presence of the underlying G to T transversion but did not find this nucleotid exchange (Figure 4), indicating that this crucial sequence of JAK2 is wt in our HL and PMBL cell lines.

Figure 4
figure4

Restriction analysis of the HL cell lines and the PMBL lines MedB-1 and Karpas1106. Gel view of a DNA 500 LabChip™ utilizing the Agilent Bioanalyzer (Agilent). (a) Using JAK2 exon 12 spanning primers we amplified the sequence (269 bp) wherein the G to T mutation occurs. (b) BsaXI digestion yielded completely cleaved genomic PCR products for all cell lines analysed. This result indicates absence of the point mutation in JAK2 exon 12 since the nucleotid exchange would abolish the BsaXI restriction site.

In this study, we analysed for the first time the SOCS-1 status of laser-microdissected HRS cells from cHL tumor tissue and a panel of HL cell lines and found that SOCS-1 is frequently mutated in cHL. At least some of these mutations result in (partly) nonfunctional hypothetical SOCS-1 proteins, which might account for high levels and accumulation of phospho-STAT5. Moreover, SOCS-1 mutations are significantly associated with nuclear accumulation of the activated STAT5. These data support the hypothesis of etiological relatedness of PMBL and cHL and suggest a common oncogenic pathway triggered by mutations in the tumor suppressor gene SOCS-1.

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Acknowledgements

We thank Michaela Buck for excellent technical assistance. This work was supported by a grant of the Deutsche Krebshilfe, Mildred Scheel Stiftung (Grant number 106367) to TFE Barth and P Möller.

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Correspondence to P Möller.

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Weniger, M., Melzner, I., Menz, C. et al. Mutations of the tumor suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation. Oncogene 25, 2679–2684 (2006). https://doi.org/10.1038/sj.onc.1209151

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Keywords

  • SOCS-1
  • Hodgkin lymphoma
  • primary mediastinal B-cell lymphoma
  • STAT5

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