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SOCS1 and SHP1 hypermethylation in mantle cell lymphoma and follicular lymphoma: implications for epigenetic activation of the Jak/STAT pathway


Mantle cell lymphoma (MCL) and follicular lymphoma (FL) are two forms of B-cell lymphoma derived from neoplastic transformation of their normal counterparts in the mantle zone and germinal center, respectively.1 MCL is characterized in virtually all cases by t(11;14), with dysregulation of the cell cycle by upregulation of cyclin D1 located at 11q13.1 On the other hand, FL is characterized by t(14;18) and upregulation of BCL-2 located at 18q21, which confers resistance to apoptosis on the malignant lymphoid cells.1

The Janus family of protein tyrosine kinase/signal transducers and activators of transcription (Jak/STAT) signaling pathway is important in the transmission of cytokine signals from cell surface to the nucleus.2 Binding of cytokines to their cognate receptors results in the dimerization of receptor complexes and activation of the Jak's, followed by phosphorylation of the cytoplasmic STATs.2 Upon phosphorylation, STATs form homo- or heterodimers, migrate to the nucleus and activate gene transcription. This Jak/STAT pathway is subject to negative regulation by PIAS members, Jak inhibitors, like the suppressor of cytokine signaling (SOCS) family proteins, and tyrosine phosphatases such as SHP1.2

The SOCS family comprises at least eight members characterized by the presence of a central src homology (SH2) domain and a conserved carboxy-terminal ‘SOCS box’.3 SOCS members, like Jak's, are cytokine inducible. SOCS1, located at 16p13.3, is a member of the SOCS family and is also known as STAT-induced STAT inhibitor. The SOCS1 gene can be induced by a multitude of cytokines including IL-1, IL-3, IL-6, LIF, EPO, GM-CSF and γ-interferon.4 Human SOCS1 is a single-exon gene encoding 211 amino acids, and lies within a CpG island spanning 2.5 kb. SOCS1 turns off cytokine signal transduction by direct interaction with Jak proteins.3

SHP1, also known as HCP, SHPTP1 and PTP1C, is a 68-kd, cytoplasmic protein tyrosine phosphatase (PTP).4 The human SHP1 gene is located on chromosome 12p13, consists of 17 exons and spans approximately 17 kb of DNA. It contains two tandem SH2 domains, a catalytic domain and a C-terminal tail of about 100 amino-acid residues.5 In contrast to the ubiquitous expression of the structurally related SHP2, SHP1 is primarily expressed in hematopoietic cells and is considered a putative tumor suppressor gene in lymphoma and leukemias, as it antagonizes the growth-promoting and oncogenic potentials of protein tyrosine kinase.4 Thus, both SOCS1 and SHP1 are possible tumor suppressors in lymphatic cells.

DNA methylation, catalyzed by DNA methyltransferase, involves the addition of a methyl group to the carbon 5 position of the cytosine ring in the CpG dinucleotide, to form methylcytosine.5 In many cancers, the CpG islands of selected genes are aberrantly methylated (hypermethylated), resulting in the repression of transcription of these genes,5 and providing an alternative mechanism of gene inactivation during tumorigenesis.

We studied cases of MCL and FL to see if hypermethylation of SOCS1 and SHP1 was contributing to lymphomagenesis by combining with upregulation of cyclin D1 in MCL and BCL-2 in FL.

Diagnosis of MCL and FL was carried out according to standard criteria. Patients were staged according to the Ann Arbor system. Immunophenotyping was performed on cryostat sections and paraffin sections with standard immunoperoxidase technique. Paraffin sections of formalin- or B5-fixed tissue were stained with hematoxylin/eosin to confirm the diagnosis of lymphoma, and examined for the expression of B- and T-cell markers. The panel of antibodies used included CD3 (Leu4, Becton Dickinson, San Jose, CA, USA), CD3 (polyclonal, Dako, Carpinteria, CA, USA), CD5 (Dako), CD10 (J5, Coulter, Miami, FL, USA), CD19 (Leu 12, Becton Dickinson), CD20 (L26, Dako), CD22 (Dako), CD23 (Novocastra, Newcastle Upon Tyne, UK) and cyclin D1 (Zymed, San Francisco, CA, USA). All 13 cases of MCL were classical variant with the phenotype of CD5+, CD10− and CD23−. The nine patients of FL belonged to FL grade II according to the WHO classification.

DNA was extracted from lymphoma cell lines (NK-YS, U266 and Raji) and frozen lymph node biopsy tissue from patients with primary MCL (n=13) and FL (n=9) by standard proteinase K digestion and phenol–chloroform extraction method.5 The methylation-specific polymerase (MSP) chain reaction, to determine gene methylation, was performed as described previously.5 Briefly, treatment of DNA with bisulfite, resulting in the conversion of unmethylated cytosine (but not methylated cytosine) to uracil, was performed with a commercially available kit (CpGenome DNA modification kit, Intergen, New York, USA) according to the manufacturer's instructions.

The sequences of the primers used for MSP and U-MSP (MSP for the unmethylated allele) of SOCS16 and SHP17 were as published previously. For SOCS1 gene methylation, primers had been designed to amplify the GC-rich region (−519 to−317) within the SOCS1 promoter, nucleotide numbers 186–335 in GenBank accession no. GI: 27486099 (NT_010393.11), subregion: complement (2121832.2123597). MSP primers (5′-IndexTermIndexTermGTTGTAGGATGGGGTCGCGGTCGC-3′ and 5′-IndexTermIndexTermCTACTAACCAAACTAAAATCCGCG-3′) were used for the detection of methylated alleles and U-MSP primers (5′-IndexTermIndexTermGTTGTAGGATGGGGTTGTGGTTGT-3′ and 5′-IndexTermIndexTermCTACTAACCAAACTAAAATCCACA-3′) for the detection of unmethylated alleles.6 For SHP1 gene methylation, primers (5′-IndexTermIndexTermGAACGTTATTATAGTATAGCGTTC-3′ and 5′-IndexTermIndexTermTCACGCATACGAACCCAAACG-3′) used for MSP had been designed for the methylated sequence of promoter region for exon 1b of the SHP1 gene (nucleotide numbers 6857–7015 in GenBank accession no. X82818) and primers (5′-IndexTermIndexTermGTGAATGTTATTATAGTATAGTGTTTGG-3′ and 5′-IndexTermIndexTermTTCACACATACA AACCCAAACAAT-3′) had been designed for the unmethylated sequence of the same region also.7

DNA from the peripheral blood of healthy donors (n=8) was used as normal controls, while methylated control DNA (CpGenome Universal Methylated DNA, Intergen) was used as positive control in all the experiments.

Apart from the methylated positive control that showed complete methylation of SOCS1, all of the eight healthy donors exhibited completely unmethylated SOCS1 and SHP1 (Figure 1) status. Methylated positive control DNA showed complete methylation by supporting amplification in M-MSP but not in U-MSP. The direct sequencing of the gel-purified MSP amplified bands of the methylated positive control DNA confirmed authenticity of SOCS1 and SHP1 amplification (results not shown). Conversely, normal control DNA showed unmethylated gene status by the presence of amplification in U-MSP but not in M-MSP. NK/T lymphoma cell line NK-YS showed aberrant hypermethylation of the SHP1 CpG island, and myeloma cell line U266 and Burkitt's lymphoma cell line Raji showed unmethylated SOCS1.

Figure 1

MSP and U-MSP of SOCS1 and SHP1 gene methylation for peripheral blood of normal controls (n=8), primary MCL (n=13) and FL (n=9) specimens. Lanes: MW – molecular weight markers; PC – methylated positive control; H2O – reagent blank and lymphoma cell lines (NK-YS, U266 and Raji).

None of the lymph node DNA samples from patients with MCL or FL showed methylation of SOCS1. On the other hand, MSP of SHP1 showed methylation in 11 of 13 (84.6%) patients with MCL and nine (100%) patients with FL (Figure 1).

Dysregulation of the Jak/STAT pathway has recently been demonstrated in lymphomas.2 For instance, activated Jak3 and STAT3/5 have been reported in cutaneous T-cell lymphoma, activated STAT3 has been shown in mycosis fungoides and activation of both STAT1 and STAT3 have been demonstrated in EBV-related lymphoproliferative diseases. Moreover, SHP1 methylation has been demonstrated in NK- and peripheral T-cell lymphomas, and methylation of SOCS1 has been demonstrated in hepatoma and myeloma.6

Our data show frequent methylation of SHP1 in B-cell lymphomas, suggesting that this gene is an important tumor suppressor in B lymphoid cells as well. The activation and deactivation of T- and B-cell immunoreceptors involves a complex signaling cascade of phosphorylation (by protein tyrosine kinase) and dephosphorylation (by PTP) of adaptor molecules or coreceptors,8 so, ultimately, we may find that the role of SHP1 methylation in lymphomagenesis extends beyond the Jak/STAT pathway.

None of our patients showed SOCS1 hypermethylation, suggesting that SOCS1 dysregulation is not important in lymphomagenesis. This notion is supported by the phenotype of SOCS1-deficient mice, which died of a myeloproliferative disease instead of a lymphoproliferative disease.3

FL is derived from germinal center B cells, and pursues an indolent clinical course. By contrast, MCL is postulated to be derived from a naïve pregerminal B cell and is a relatively aggressive lymphoma.1 Despite these clinicopathological differences, we show frequent methylation of SHP1 in both FL and MCL. Together with the finding of frequent methylation of SHP1 in NK- and peripheral T-cell lymphomas,7 these results suggest that SHP1 dysregulation and, possibly, constitutive activation of the Jak/STAT signaling pathway is fundamental to the pathogenesis of lymphomas with a wide range of origin, histology and clinical aggressiveness.1

Genetic aberrations that may cooperate with upregulated cyclin D1 in lymphomagenesis have been demonstrated in MCL. For instance, alterations in cell cycle control genes such as downregulation of RB and deletion or hypermethylation of p15 and p16 have been identified in patients with MCL. Frequent inactivating mutations of ATM in MCL have also been demonstrated. Our finding of frequent SHP1 hypermethylation, possibly resulting in constitutive, epigenetic activation of the Jak/STAT pathway, illustrates another genetic alteration that can act, in addition to cyclin D1, in MCL lymphomagenesis. These findings concur with those in transgenic mice models, in which overexpression of cyclin D1 alone was insufficient, and additional overexpression of c-myc was required, for lymphomagenesis.1

Similarly, in BCL2 transgenic mice, only a small fraction of mice develop lymphomas. That small fraction shows a high-grade phenotype only after prolonged latency, and this involves additional genetic alteration in c-MYC. We hypothesize that SHP1 hypermethylation may also collaborate with the upregulation of BCL2 in lymphomagenesis of FL.1 Whether concurrent BCL2 overexpression and SHP1 inactivation might give rise to low-grade lymphoma in mice remains to be tested.

In summary, frequent methylation of SHP1, but not SOCS1, occurs in both mantle cell and FLs, suggesting that SHP1 inactivation might be an important secondary event in lymphomagenesis in addition to the upregulation of cyclin D1 and BCL2 in mantle cell and FLs, respectively. Therefore, demethylating agents and histone deacetylase inhibitors are potentially useful therapeutic agents. The effect of hypermethylation of SHP1 on the Jak/STAT signaling pathways in lymphomas should be explored, and other forms of Jak/STAT deregulation, such as mutational inactivation of SOC1, should also be investigated.


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We express our sincere thanks to Dr Junjiro Tsuchiyama for the NK-YS cell line and all medical and nursing staff in the Department of Medicine, Queen Mary Hospital for the provision of expert medical care.

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Correspondence to C S Chim or G Srivastava.

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