Epigenetic Silencing of Stk39 in B-Cell Lymphoma Inhibits Apoptosis from Genotoxic Stress
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090091
ISSN1525-2191
AutoresCynthia E. Balatoni, David W. Dawson, Jane Suh, Mara H. Sherman, Grant Sanders, Jason Hong, Matthew J. Frank, Cindy S. Malone, Jonathan W. Said, Michael A. Teitell,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoB-cell lymphomas, the most frequent human immune system malignancies, often contain dysregulated TCL1 oncogene expression. TCL1 transgenic (TCL1-tg) mice develop a spectrum of B-cell malignancies, supporting an oncogenic role for TCL1 in B cells. Our prior global survey of DNA methylation patterns in TCL1-tg B-cell lymphomas identified many lymphoma-specific candidate hypermethylated genes, including Stk39. The Stk39 encoded protein, sterile 20-like-related proline-alanine-rich kinase (SPAK), regulates cell stress responses, and microarray studies identified reduced SPAK expression in metastatic prostate and treatment-resistant breast cancers, suggesting that its loss may have a role in cancer progression. Here we identified DNA hypermethylation and SPAK silencing in TCL1-tg B-cell lymphomas and SPAK silencing without DNA methylation in multiple subtypes of human B-cell lymphomas. SPAK knockdown by shRNA protected B cells from caspase-dependent apoptosis induced by DNA double-strand breaks but not apoptosis in response to osmotic or oxidative cell stressors. Caspase 3 activation by cleavage was impaired with SPAK repression in DNA damaged B cells. Interestingly, c-Jun NH2-terminal kinase is potentially activated by SPAK and pharmacological inhibition of c-Jun NH2-terminal kinase in SPAK-expressing B cells recapitulated the cell-protective phenotype of SPAK knockdown. Taken together, these data indicate that SPAK loss in B-cell lymphomas promotes increased cell survival with DNA damage and provides a potential mechanism for increased resistance to genotoxic stress in cancer. B-cell lymphomas, the most frequent human immune system malignancies, often contain dysregulated TCL1 oncogene expression. TCL1 transgenic (TCL1-tg) mice develop a spectrum of B-cell malignancies, supporting an oncogenic role for TCL1 in B cells. Our prior global survey of DNA methylation patterns in TCL1-tg B-cell lymphomas identified many lymphoma-specific candidate hypermethylated genes, including Stk39. The Stk39 encoded protein, sterile 20-like-related proline-alanine-rich kinase (SPAK), regulates cell stress responses, and microarray studies identified reduced SPAK expression in metastatic prostate and treatment-resistant breast cancers, suggesting that its loss may have a role in cancer progression. Here we identified DNA hypermethylation and SPAK silencing in TCL1-tg B-cell lymphomas and SPAK silencing without DNA methylation in multiple subtypes of human B-cell lymphomas. SPAK knockdown by shRNA protected B cells from caspase-dependent apoptosis induced by DNA double-strand breaks but not apoptosis in response to osmotic or oxidative cell stressors. Caspase 3 activation by cleavage was impaired with SPAK repression in DNA damaged B cells. Interestingly, c-Jun NH2-terminal kinase is potentially activated by SPAK and pharmacological inhibition of c-Jun NH2-terminal kinase in SPAK-expressing B cells recapitulated the cell-protective phenotype of SPAK knockdown. Taken together, these data indicate that SPAK loss in B-cell lymphomas promotes increased cell survival with DNA damage and provides a potential mechanism for increased resistance to genotoxic stress in cancer. Leukemias and lymphomas arise by genetic and epigenetic alterations of previously healthy lymphocytes. B-cell lymphoma is the most frequent lymphocyte malignancy, with certain tumor subtypes characterized by recurring genetic alterations that include reciprocal chromosome translocations between IG loci and CCND1, BCL2, MYC, and BCL6.1Kuppers R Mechanisms of B-cell lymphoma pathogenesis.Nat Rev Cancer. 2005; 5: 251-262Crossref PubMed Scopus (676) Google Scholar These rearrangements place tumor-promoting genes under the control of IG rather than endogenous regulatory elements, leading to their dysregulated expression. Epigenetic changes, including DNA hypermethylation, aberrant histone modifications, and altered microRNA expression are also linked to B-cell transformation.2Calin GA Dumitru CD Shimizu M Bichi R Zupo S Noch E Aldler H Rattan S Keating M Rai K Rassenti L Kipps T Negrini M Bullrich F Croce CM Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia.Proc Natl Acad Sci USA. 2002; 99: 15524-15529Crossref PubMed Scopus (4196) Google Scholar, 3Costello JF Fruhwald MC Smiraglia DJ Rush LJ Robertson GP Gao X Wright FA Feramisco JD Peltomaki P Lang JC Schuller DE Yu L Bloomfield CD Caligiuri MA Yates A Nishikawa R Su Huang H Petrelli NJ Zhang X O'Dorisio MS Held WA Cavenee WK Plass C Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.Nat Genet. 2000; 24: 132-138Crossref PubMed Scopus (1163) Google Scholar, 4Shilatifard A Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression.Annu Rev Biochem. 2006; 75: 243-269Crossref PubMed Scopus (885) Google Scholar In particular, DNA hypermethylation promotes chromatin compaction and gene silencing, with consistent repression of specific tumor suppressor genes associated with multiple types of cancer, including subtypes of B-cell leukemia and lymphoma.5French SW Dawson DW Miner MD Doerr JR Malone CS Wall R Teitell MA DNA methylation profiling: a new tool for evaluating hematologic malignancies.Clin Immunol. 2002; 103: 217-230Crossref PubMed Scopus (10) Google Scholar Aberrant expression of the TCL1 oncogene, first identified from rearrangements with TCR loci in T-cell prolymphocytic leukemia, also occurs frequently in mature B-cell leukemias and lymphomas, although not by gene rearrangement [reviewed in6Teitell MA The TCL1 family of oncoproteins: co-activators of transformation.Nat Rev Cancer. 2005; 5: 640-648Crossref PubMed Scopus (83) Google Scholar]. A causative role for ectopic TCL1 expression in lymphocyte transformation is supported by three different TCL1 transgenic (TCL1-tg) mouse models that develop mature B- and T-cell malignancies.6Teitell MA The TCL1 family of oncoproteins: co-activators of transformation.Nat Rev Cancer. 2005; 5: 640-648Crossref PubMed Scopus (83) Google Scholar, 7Hoyer KK French SW Turner DE Nguyen MT Renard M Malone CS Knoetig S Qi CF Su TT Cheroutre H Wall R Rawlings DJ Morse 3rd, HC Teitell MA Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma.Proc Natl Acad Sci USA. 2002; 99: 14392-14397Crossref PubMed Scopus (99) Google Scholar TCL1-tg mice and patients with dysregulated TCL1 expression exhibit polyclonal lymphocyte hyperplasia and a long delay to tumor formation, suggesting that additional genetic or epigenetic lesions are required for transformation.7Hoyer KK French SW Turner DE Nguyen MT Renard M Malone CS Knoetig S Qi CF Su TT Cheroutre H Wall R Rawlings DJ Morse 3rd, HC Teitell MA Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma.Proc Natl Acad Sci USA. 2002; 99: 14392-14397Crossref PubMed Scopus (99) Google Scholar Supporting this idea, our prior work identified companion chromosomal translocations, aneuploidy, and Myc overexpression in TCL1-tg B-cell lymphomas.8Shen RR Ferguson DO Renard M Hoyer KK Kim U Hao X Alt FW Roeder RG Morse 3rd, HC Teitell MA Dysregulated TCL1 requires the germinal center and genome instability for mature B-cell transformation.Blood. 2006; 108: 1991-1998Crossref PubMed Scopus (14) Google Scholar To elucidate epigenetic alterations that accompany and may facilitate transformation or lymphoma progression, unbiased genome-wide DNA methylation analysis of more than 2000 genetic loci using restriction landmark genomic scanning (RLGS) was performed and demonstrated reproducible nonrandom patterns of aberrant DNA methylation in TCL1-tg B-cell lymphomas compared with wild-type or nontransformed TCL1-tg B cells.9Dawson DW Hong JS Shen RR French SW Troke JJ Wu YZ Chen SS Gui D Regelson M Marahrens Y Morse 3rd, HC Said J Plass C Teitell MA Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas.Oncogene. 2007; 26: 4243-4252Crossref PubMed Scopus (35) Google Scholar This RLGS survey detected tumor-associated hypermethylation of 115 candidate loci including two loci belonging to serine/threonine protein kinase 39 (Stk39). STK39 encodes STE20 (sterile 20-like)-related proline-alanine-rich kinase (SPAK), one of two members of the germinal center kinase VI subgroup within the STE20 kinase family.10Delpire E Gagnon KB SPAK and OSR1: STE20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells.Biochem J. 2008; 409: 321-331Crossref PubMed Scopus (173) Google Scholar In general, mammalian STE20 kinases are implicated in osmotic stress signaling. SPAK is a 60-kDa protein that contains an N-terminal PAPA box of unknown function, a serine/threonine kinase domain, a putative nuclear localization signal, and a C-terminal region that binds to SPAK target proteins. SPAK is ubiquitously expressed and modulates ion and fluid homeostasis through interactions with and direct phosphorylation of the cation chloride cotransporters KCC3, NKCC1, and NKCC2.11Piechotta K Lu J Delpire E Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1).J Biol Chem. 2002; 277: 50812-50819Crossref PubMed Scopus (304) Google Scholar Interestingly, SPAK activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway independent of its target binding C-terminal region. c-Jun NH2-terminal kinase (JNK) MAPK may also be a direct SPAK target, implying an independent SPAK function as an upstream MAP4K.12Johnston AM Naselli G Gonez LJ Martin RM Harrison LC DeAizpurua HJ SPAK, a STE20/SPS1-related kinase that activates the p38 pathway.Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (115) Google Scholar Expression microarray studies have identified a correlation between reduced STK39 expression in prostate cancer metastasis and in tumor relapse and resistance to certain treatments in breast cancer.13Cleator S Tsimelzon A Ashworth A Dowsett M Dexter T Powles T Hilsenbeck S Wong H Osborne CK O'Connell P Chang JC Gene expression patterns for doxorubicin (Adriamycin) and cyclophosphamide (Cytoxan) (AC) response and resistance.Breast Cancer Res Treat. 2006; 95: 229-233Crossref PubMed Scopus (80) Google Scholar, 14Hendriksen PJ Dits NF Kokame K Veldhoven A van Weerden WM Bangma CH Trapman J Jenster G Evolution of the androgen receptor pathway during progression of prostate cancer.Cancer Res. 2006; 66: 5012-5020Crossref PubMed Scopus (181) Google Scholar However, these studies did not independently validate these microarray results nor did they correlate the loss of STK39 with tumor progression. Likewise, the role of SPAK in B cells or in B-cell tumors is not known, prompting us to further investigate Stk39 and SPAK. Here we show that SPAK expression is repressed or silenced in TCL1-tg B-cell tumors and that reduced expression is accompanied by Stk39 DNA hypermethylation. Moreover, loss of SPAK expression was observed among several primary human B-cell lymphoma subtypes, suggesting that SPAK loss may be a shared feature of both human disease and the Eμ-B29-hTCL1 mouse model of lymphoma. We show that decreased SPAK expression protected B cells from apoptosis induced by DNA double-strand breaks (DSBs) through a mechanism dependent on caspase activation. This protection was independent of known SPAK downstream effectors, including p38 and the cation chloride cotransporter proteins. Loss of SPAK expression increases the survival of B cells with DNA DSBs, which can occur with specific genotoxic stressors or during a T-cell-dependent germinal center (GC) reaction. By increasing survival, impaired signaling from SPAK loss may promote malignant progression by abrogating the elimination of DNA-damaged B cells and possibly other cell types with DNA damage as well. TCL1-tg B-cell tumors were from spleens of 9- to 16-month-old mice.7Hoyer KK French SW Turner DE Nguyen MT Renard M Malone CS Knoetig S Qi CF Su TT Cheroutre H Wall R Rawlings DJ Morse 3rd, HC Teitell MA Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma.Proc Natl Acad Sci USA. 2002; 99: 14392-14397Crossref PubMed Scopus (99) Google Scholar Tumor sample Burkitt-like lymphoma (BLL) 1a is a daughter tumor that arose at 6 weeks from adoptive transfer of a primary TCL1-tg B-cell tumor (BLL1) into a 4-month-old syngeneic mouse. All other TCL1-tg B-cell tumors were from the primary source mice. Nalm6 and BCBL1 B cells were grown in RPMI 1640 and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum. Lymphomas were examined by histology and immunophenotype, classified according to the World Health Organization system, and retrieved from the files of the UCLA Department of Pathology.15Harris NL Jaffe ES Diebold J Flandrin G Muller-Hermelink HK Vardiman J Lister TA Bloomfield CD The World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues. Report of the Clinical Advisory Committee meeting, Airlie House, Virginia, November, 1997.Ann Oncol. 1999; 10: 1419-1432Crossref PubMed Scopus (486) Google Scholar SPAK antiserum (L. Harrison, Parkville, VIC, Australia) was used at 1:1000 dilution and tumor cell staining was scored on a 0 to 4+ scale in increments of 25% by two pathologists.16Said JW Hoyer KK French SW Rosenfelt L Garcia-Lloret M Koh PJ Cheng TC Sulur GG Pinkus GS Kuehl WM Rawlings DJ Wall R Teitell MA TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.Lab Invest. 2001; 81: 555-564Crossref PubMed Scopus (76) Google Scholar SPAK expression was detected using the Lab Vision Ultravision ONE Polymer Immunohistochemistry Detection System (Fisher Scientific, Pittsburgh, PA) with horseradish peroxidase (HRP) and DAB Chromogen and a hematoxylin counterstain. In brief, after deparaffinization, rehydration, and antigen retrieval using a vegetable steamer, slides were blocked and incubated overnight at 4°C with rabbit antisera specific for SPAK. Genomic DNA was isolated from fresh-frozen TCL1-tg B-cell lymphomas or control spleens using TRIzol (Invitrogen, Carlsbad, CA). PCR reactions of 40 cycles were performed on sodium bisulfite-treated genomic DNA, which was prepared as described previously.17Doerr JR Malone CS Fike FM Gordon MS Soghomonian SV Thomas RK Tao Q Murray PG Diehl V Teitell MA Wall R Patterned CpG methylation of silenced B cell gene promoters in classical Hodgkin lymphoma-derived and primary effusion lymphoma cell lines.J Mol Biol. 2005; 350: 631-640Crossref PubMed Scopus (52) Google Scholar Agarose gel-purified amplicons were cloned into the pCR2.1-TOPO vector (Invitrogen) with clones isolated and sequenced. PCR primers for bisulfite-converted gDNA were Stk39 forward (5′-AAGT-GTTTTTGAATAAAAGAGAAAGT-3′) and Stk39 reverse (5′-AAAAAACCCCCCTAAAA-CTCC-3′). The PCR-amplified product spanned the region −229 to +73 upstream of the major Stk39 transcriptional start site and was 302 nucleotides long (). RNA was extracted using TRIzol and first-strand cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen). SYBR green quantitative PCR was performed using an Applied Biosystems 7700 sequence detector. Samples were analyzed for Stk39 and the housekeeping gene 36b4 (Rplp0). The primers for human and mouse STK39 and 36B4 were as follows: hSTK39 forward (5′-GCCATCCCAACGTAGTGACC-3′), hSTK39 reverse (5′-TGTTCT-CCTCGGTTGACAATGTA-3′), h36B4 forward (5′-CCCGCTGCTGAACATGCT-3′), h36B4 reverse (5′-TCGAACACCTGCTGGATGAC-3′), mSTK39 forward (5′-GCGTAGCCATAAAG-CGGATC-3′), mSTK39 reverse (5′-TACGTTGGGATGGCTGCATT-3′), m36B4 forward (5′-AGATGCAGCAGATCCGCAT-3′), and m36B4 reverse (5′-GTTCTTGCCCATCAGCACC-3′). Each sample was examined in triplicate with a two-tailed t-test to determine statistical significance. Mouse B cells were isolated using an AUTOMACS Cell Sorter (Miltenyi Biotech, Auburn, CA). Incubation with CD4-phycoerythrin (PE), CD8-PE, and Gr1-PE antibodies (BD Pharmingen, San Diego, CA) was followed with anti-PE microbeads (Miltenyi Biotech). B cells were collected by negative selection, with all other cells eluted as the non-B-cell fraction. Tonsillar B cells were isolated and sorted into naive, memory, and germinal center subpopulations, as described previously.16Said JW Hoyer KK French SW Rosenfelt L Garcia-Lloret M Koh PJ Cheng TC Sulur GG Pinkus GS Kuehl WM Rawlings DJ Wall R Teitell MA TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.Lab Invest. 2001; 81: 555-564Crossref PubMed Scopus (76) Google Scholar Three STK39 shRNA vectors, RNAi1 (5′-GGTGGATGGTCACGATGTA-3′), RNAi2 (5′-ATTCAAGCCATGAGTCAGT-3′), and RNAi3 (5′-GAGCAGCGCCTTATCACAA-3′),18Li Y Hu J Vita R Sun B Tabata H Altman A SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway.EMBO J. 2004; 23: 1112-1122Crossref PubMed Scopus (74) Google Scholar were generated by cloning DNA oligomers (Dharmacon, Lafayette, CO) into the H1 promoter-driven retroviral plasmid pQSuper (S. Smale, UCLA). To generate retroviruses, HEK293T fibroblasts were transfected overnight using Lipofectamine 2000 (Invitrogen) with 12 μg of each shRNA construct, 4 μg of murine leukemia virus gag/pol pHIT60, and 2 μg of vesicular stomatitis virus glycoprotein. Retrovirus-containing media was collected every 12 hours for 72 hours, and 8 μg/ml Polybrene was added. B-cell lines (1 × 105/ml) were spin-infected at 2500 rpm for 1 hour at 30°C and resuspended in fresh media. Two days later, cells were placed on 1 μg/ml puromycin selection. Cells were washed twice in 1× PBS, pH 7.4, and lysed in Triton X-100 1× cell lysis buffer (9803; Cell Signaling Technology, Danvers, MA) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride. The 1× lysis buffer contained 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, and 1 μg/ml leupeptin. Protein lysates (40 μg) were resolved by 3% stacking/8% resolving SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, incubated for 1 hour with 5% milk Tris-buffered saline-Tween 20 and overnight with primary antibodies in 5% BSA. Antibodies from Cell Signaling Technology were the following: SPAK (1:1000) (2281), OSR1 (1:1000) (3729), caspase 3 (1:1000) (9662), phospho-SAPK/JNK (Thr183/Tyr185) (1:500) (9255), phospho-p53 (Ser15) (1:1000) (9284), and anti-mouse-IgG-HRP (1:5000) (7076). Antibodies from Sigma-Aldrich (St. Louis, MO) were β-tubulin (1:1000) (T-4026) and actin (1:20,000) (A-2066). Anti-rabbit-HRP (1:5000) (711-035-152) was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). ECL reagent (RPN 2209; GE Healthcare, Piscataway, NJ) was used for chemiluminescent detection. Densitometry was performed using SynGene Analysis software (SynGene, Upland, CA). Nalm6 or BCBL1 B cells were seeded at 5 × 105 cells/ml and preincubated for 1 hour with Q-VD-OPH (MP Biomedicals, Solon, OH), furosemide or bumetanide (Sigma- Aldrich), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), or U0126 (mitogen-activated protein kinase kinase-1/2 inhibitor) (Calbiochem, San Diego, CA). Drug concentrations and incubation times are provided in the figure legends for each experiment. Cells were exposed to ionizing radiation (IR) using a Cs-137 Mark I Irradiator, exposed to UV-B using a Spectrolinker XL-1000 crosslinker (Spectronics Corporation, Westbury, NY), or incubated with etoposide, hydrogen peroxide, or sorbitol (Sigma-Aldrich). Apoptosis was measured using annexin V-FITC and propidium iodide permeability (BD Pharmingen). Vital staining was quantified using a Becton Dickinson FACScan analytic flow cytometer, and data were analyzed using CellQuest software (BD Biosciences, San Jose, CA). Each flow cytometry sample was examined in triplicate with a two-tailed t-test to determine statistical significance. Previously, RLGS profiling of genome-wide DNA methylation patterns in TCL1-tg B-cell tumors identified Stk39 as a candidate hypermethylated gene.9Dawson DW Hong JS Shen RR French SW Troke JJ Wu YZ Chen SS Gui D Regelson M Marahrens Y Morse 3rd, HC Said J Plass C Teitell MA Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas.Oncogene. 2007; 26: 4243-4252Crossref PubMed Scopus (35) Google ScholarStk39 was chosen from the list of candidate hypermethylated genes for further study because of its potential loss in cancer progression and its known role in hyperosmotic cell stress responses.11Piechotta K Lu J Delpire E Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1).J Biol Chem. 2002; 277: 50812-50819Crossref PubMed Scopus (304) Google Scholar Supporting this reasoning, hyperosmotic stress induces cell cycle arrest, mitotic catastrophe, and apoptosis of colonic and pancreatic cancer cells,19Tao GZ Rott LS Lowe AW Omary MB Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation.J Biol Chem. 2002; 277: 19295-19303Crossref PubMed Scopus (24) Google Scholar suggesting a potential role for Stk39 silencing in lymphoma progression. Genomic bisulfite sequencing of a 255-bp stretch containing 19 CpG sites in a Stk39 promoter CpG island was performed to assess the extent of DNA methylation and validate the RLGS data (Figure 1A). This region was selected for study because it was part of a larger CpG island that extended into the transcribed region of the gene and because bisulfite sequencing primers could be optimally designed at this site. Only 0.4 CpG sites per clone (2%) were methylated in wild-type (WT1) spleen cells, whereas averages of 9.1 (48%) and 8.4 (44%) CpG sites per clone were methylated in BLL1 and BLL3 mouse tumors, respectively. These data validate RLGS results and demonstrate that the Stk39 promoter is frequently hypermethylated in B-cell lymphomas arising in TCL1-tg mice. To determine whether promoter methylation correlated with STK39 expression, the steady-state mRNA level was analyzed by quantitative PCR. When compared with Stk39 expression in wild-type and premalignant TCL1-tg spleens, tumors with Stk39 DNA methylation showed an average 5.6-fold reduction in Stk39 expression (1.73 ± 0.99 vs. 0.31 ± 0.24, P = 0.003) (Figure 1B). In contrast, the average Stk39 expression between wild-type and premalignant TCL1-tg spleens was not statistically different (2.10 ± 1.22 vs. 1.37 ± 0.64, P = 0.23). SPAK protein expression was also determined in splenocytes from wild-type, premalignant TCL1-tg, and tumorigenic TCL1-tg mice. Protein lysates from the eight original tumor samples in Figure 1B were unavailable, so newly identified TCL1-tg B-cell tumors confirmed by histology and flow cytometry were used, as described previously.7Hoyer KK French SW Turner DE Nguyen MT Renard M Malone CS Knoetig S Qi CF Su TT Cheroutre H Wall R Rawlings DJ Morse 3rd, HC Teitell MA Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma.Proc Natl Acad Sci USA. 2002; 99: 14392-14397Crossref PubMed Scopus (99) Google Scholar SPAK was expressed in both sorted B and non-B cells from 2 month-old wild-type (WT5) and 10-month-old premalignant TCL1-tg (Tg5) spleens (Figure 1C). In contrast, SPAK was not detected in cells from an 18-month-old TCL1-tg splenic tumor (diffuse large B-cell lymphoma [DLBCL] 3) and was markedly repressed in malignant B and non-B cells from a 19-month-old TCL1-tg tumor (BLL4). Four additional splenic TCL1-tg B-cell lymphomas showed a similar loss of SPAK expression (Supplemental Figure S1, see ). Combined, these data indicate that SPAK expression is repressed in TCL1-tg B-cell lymphomas that also show promoter region DNA hypermethylation. To evaluate whether SPAK repression also occurs in human tumors, a panel of 42 paraffin-embedded formalin-fixed human B-lymphoma samples were examined for SPAK expression by immunohistochemistry (IHC). Stained sections of hyperplastic tonsil showed stronger SPAK expression in follicular center cells compared with mantle and interfollicular zone cells (Figure 2A). In addition, B cells from tonsil were sorted into naive, GC, and memory subsets, with a Western blot showing SPAK expression throughout mature, nonmalignant B-cell development (Figure 2B). SPAK expression was determined in Burkitt lymphoma (n = 4), DLBCL (n = 11), follicular lymphoma (n = 11), marginal zone lymphoma (n = 6), mantle cell lymphoma (n = 5), and small lymphocytic lymphoma (n = 5). The range of SPAK expression was wide, as shown by a strongly positive marginal zone lymphoma sample (Figure 2C) and a negative small lymphocytic lymphoma sample (Figure 2D). Samples were scored by two pathologists for the percentage of SPAK-expressing tumor cells on a scale ranging from 0 (negative) to 4+ (75 to 100% positive tumor cells) in increments of 25%. For comparison, tonsil shows between 50 to 100% (3+ to 4+) SPAK-positive cell staining as a reference. All of the lymphoma subtypes, with the exception of Burkitt lymphoma, had at least one SPAK negative (0) sample (Figure 2E). All five small lymphocytic lymphoma samples had <50% SPAK-positive tumor cells. Average SPAK expression was highest for the Burkitt lymphoma samples (3.0 ± 0.8), followed by DLBCL (2.4 ± 1.5), marginal zone lymphoma (2.3 ± 1.6), follicular lymphoma (1.4 ± 1.6), mantle cell lymphoma (1.2 ± 1.3), and small lymphocytic lymphoma (1.2 ± 0.8). The loss of SPAK expression may reflect the progression from SPAK-positive B cells, such as in the tonsil, to SPAK-negative tumor cells over time. Samples that have an intermediate amount of tumor cell staining may reflect an intermediate stage in tumor evolution and a heterogeneous population of normal and lymphoma B cells, but this theory will require further investigation. Overall, these results indicate that 34 of 42 (81%) B lymphoma samples contained at least 25% tumor cells lacking SPAK expression (Figure 2F), suggesting that SPAK repression may be a progressive feature of murine and human B-cell lymphomas. STK39 showed sparse DNA methylation in just 2 of 20 (10%) DLBCL and 0 of 20 follicular lymphoma samples, as determined by methylation-specific PCR and sample-selected genomic bisulfite sequencing (data not shown). This lack of DNA methylation, coupled with progressive SPAK silencing, suggests that there are distinct pathways for human B-cell lymphomas and mouse TCL1-tg B-cell lymphomas in repressing SPAK expression. Consistent with this result, loss of SPAK expression in metastatic prostate and treatment-resistant breast cancers has not been linked to aberrant DNA methylation,13Cleator S Tsimelzon A Ashworth A Dowsett M Dexter T Powles T Hilsenbeck S Wong H Osborne CK O'Connell P Chang JC Gene expression patterns for doxorubicin (Adriamycin) and cyclophosphamide (Cytoxan) (AC) response and resistance.Breast Cancer Res Treat. 2006; 95: 229-233Crossref PubMed Scopus (80) Google Scholar, 14Hendriksen PJ Dits NF Kokame K Veldhoven A van Weerden WM Bangma CH Trapman J Jenster G Evolution of the androgen receptor pathway during progression of prostate cancer.Cancer Res. 2006; 66: 5012-5020Crossref PubMed Scopus (181) Google Scholar suggesting another silencing mechanism in humans that requires further investigation. To investigate a role for repressed SPAK expression in B-cell lymphoma, nonoverlapping STK39-targeted shRNA retroviral constructs were tested, with one knockdown construct generated against a previously published target region of STK39 (RNAi2)18Li Y Hu J Vita R Sun B Tabata H Altman A SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway.EMBO J. 2004; 23: 1112-1122Crossref PubMed Scopus (74) Google Scholar and the other constructed using a newly selected targeted region (RNAi1). Virus was generated and used to infect human Nalm6 B cells. Nalm6 cells were chosen because they expressed SPAK and tolerated retroviral infection to generate stable STK39 knockdown B cells, whereas unstimulated primary B cells died in culture by 7 days. At 2 weeks after infection STK39 expression was reduced by 85% (P = 0.02) with RNAi1 or 53% (P = 0.05) with RNAi2 compared with that in parent cells (Figure 3A). SPAK protein expression with RNAi1 infection was also markedly reduced compared with that in control cells (Figure 3B). Despite 74% sequence identity between SPAK and the only other STE-20 subfamily member, OSR1, RNAi1 showed strong SPAK specificity (Figure 3B). Because of its strong specificity and superior SPAK knockdown compared with a prior published SPAK knockdown construct (RNAi2),18Li Y Hu J Vita R Sun B Tabata H Altman A SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway.EMBO J. 2004; 23: 1112-1122Crossref PubMed Scopus (74) Google Scholar RNAi1 was used for all subsequent studies. Resting RNAi1 Nalm6 cells seemed unaffected in culture and showed no difference in viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays (data not shown). SPAK has been shown to activate p38 and possibly JNK MAPK pathways in response to osmotic stress,12Johnston AM Naselli G Gonez LJ Martin RM Harrison LC DeAizpurua HJ SPAK, a STE20/SPS1-related kinase that activates the p38 pathway.Oncogene. 2000; 19: 4290-4297Crossref PubMed Scopus (115) Google Scholar, 18Li Y Hu J Vita R Sun B Tabata H Altman A SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway.EMBO J. 2004; 23: 1112-1122Crossref PubMed Scopus (74) Google Scholar, 20Piechotta K Garbarini N
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