Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1+, while reactive lymphoid hyperplasia (n = 12) was RanGAP1+ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin’s lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.
References
[1]
Schneider RJ, Seibert K, Passe S, Little C, Gee T et al. (1980) Prognostic significance of serum lactate dehydrogenase in malignant lymphoma. Cancer 46: 139-143. doi:10.1002/1097-0142(19800701)46:1. PubMed: 6992974.
[2]
McAdam B, Smith T, Love WC, Murphy M, Daly PA (1993) Lactate dehydrogenase levels during MACOP-B chemotherapy for non-Hodgkin's lymphoma. Med Oncol Tumor Pharmacother 10: 95-101. PubMed: 7505374.
[3]
Sarris AH, Majlis A, Dimopoulos MA, Younes A, Swann F et al. (1995) Rising serum lactate dehydrogenase often caused by granulocyte-or Granulocyte-macrophage colony stimulating factor and not tumor progression in patients with lymphoma or myeloma. Leuk Lymphoma 17: 473-477. doi:10.3109/10428199509056860. PubMed: 7549840.
[4]
Hagberg H, Killander A, Simonsson B (1983) Serum beta 2-microglobulin in malignant lymphoma. Cancer 51: 2220-2225. doi:10.1002/1097-0142(19830615)51:12. PubMed: 6189572.
[5]
Krejsek J, Slezák R, Kopecky O, Derner V, Andrys C (1997) Elevation of serum soluble intercellular adhesion molecule-1 (sICAM-1) and beta-2-microglobulin in Sjogren's syndrome. Clin Rheumatol 16: 149-153. doi:10.1007/BF02247843. PubMed: 9093796.
[6]
Mulaomerovi？ A, Halilbasi？ A, Cickusi？ E, Zavasnik-Bergant T, Begi？ L et al. (2007) Cystatin C as a potential marker for relapse in patients with non-Hodgkin B-cell lymphoma. Cancer Lett 248: 192-197. doi:10.1016/j.canlet.2006.07.004. PubMed: 16945481.
[7]
Terol MJ, Tormo M, Martinez-Climent JA, Marugan I, Benet I et al. (2003) Soluble intercellular adhesion molecule-1 (s-ICAM-1/s-CD54) in diffuse large B-cell lymphoma: association with clinical characteristics and outcome. Ann Oncol 14: 467-474. doi:10.1093/annonc/mdg057. PubMed: 12598355.
[8]
Niitsu N, Sasaki K, Umeda M (1999) A high serum soluble Fas/APO-1 level is associated with a poor outcome of aggressive non-Hodgkin's lymphoma. Leukemia 13: 1434-1440. doi:10.1038/sj/leu/2401502. PubMed: 10482996.
[9]
Goto N, Tsurumi H, Takemura M, Hara T, Sawada M et al. (2006) Serum-soluble tumor necrosis factor receptor 2 (sTNF-R2) level determines clinical outcome in patients with aggressive non-Hodgkin's lymphoma. Eur J Haematol 77: 217-225. doi:10.1111/j.1600-0609.2006.00702.x. PubMed: 16856931.
[10]
Goto H, Tsurumi H, Takemura M, Ino-Shimomura Y, Kasahara S et al. (2005) Serum-soluble interleukin-2 receptor (sIL-2R) level determines clinical outcome in patients with aggressive non-Hodgkin's lymphoma: in combination with the International Prognostic Index. J Cancer Res Clin Oncol 131: 73-79. doi:10.1007/s00432-004-0600-9. PubMed: 15503137.
[11]
Niitsu N, Okabe-Kado J, Okamoto M, Takagi T, Yoshida T et al. (2001) Serum nm23-H1 protein as a prognostic factor in aggressive non-Hodgkin lymphoma. Blood 97: 1202-1210. doi:10.1182/blood.V97.5.1202. PubMed: 11222361.
[12]
Niitsu N, Iijima K (2002) High serum soluble CD44 is correlated with a poor outcome of aggressive non-Hodgkin's lymphoma. Leuk Res 26: 241-248. doi:10.1016/S0145-2126(01)00122-9. PubMed: 11792412.
[13]
Cimerman N, Brguljan PM, Krasovec M, Suskovic S, Kos J (2000) Serum cystatin C, a potent inhibitor of cysteine proteinases, is elevated in asthmatic patients. Clin Chim Acta 300: 83-95. doi:10.1016/S0009-8981(00)00298-9. PubMed: 10958865.
[14]
Konno R, Takano T, Sato S, Yajima A (2000) Serum soluble fas level as a prognostic factor in patients with gynecological malignancies. Clin Cancer Res 6: 3576-3580. PubMed: 10999747.
[15]
Rentzos M, Michalopoulou M, Nikolaou C, Cambouri C, Rombos A et al. (2005) The role of soluble intercellular adhesion molecules in neurodegenerative disorders. J Neurol Sci 228: 129-135. doi:10.1016/j.jns.2004.11.001. PubMed: 15694193.
[16]
Chang KC, Huang GC, Jones D, Tsao CJ, Lee JY et al. (2004) Distribution and prognosis of WHO lymphoma subtypes in Taiwan reveals a low incidence of germinal-center derived tumors. Leuk Lymphoma 45: 1375-1384. doi:10.1080/10428194042000198849. PubMed: 15359636.
[17]
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA et al. (2008) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: International Agency for Research on Cancer.
[18]
Coiffier B (2003) Immunochemotherapy: the new standard in aggressive non-Hodgkin's lymphoma in the elderly. Semin Oncol 30: 21-27. doi:10.1053/sonc.2003.50029. PubMed: 12652461.
[19]
Neitzel H (1986) A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet 73: 320-326. doi:10.1007/BF00279094. PubMed: 3017841.
[20]
Chang Y, Tung CH, Huang YT, Lu J, Chen JY et al. (1999) Requirement for cell-to-cell contact in Epstein-Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes. J Virol 73: 8857-8866. PubMed: 10482644.
[21]
Chang WC, Chou CK, Tsou CC, Li SH, Chen CH et al. (2010) Comparative proteomic analysis of proteins involved in the tumorigenic process of seminal vesicle carcinoma in transgenic mice. International J of Proteomics 2010: 1-14 PubMed: 22084680.
[22]
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26: 1367-1372. doi:10.1038/nbt.1511. PubMed: 19029910.
[23]
Chang KC, Chen PC, Chen YP, Chang Y, Su IJ (2011) Dominant expression of survival signals of endoplasmic reticulum stress response in Hodgkin lymphoma. Cancer Sci 102: 275-281. doi:10.1111/j.1349-7006.2010.01765.x. PubMed: 21062387.
[24]
Yeh YM, Chang KC, Chen YP, Kao LY, Tsai HP et al. (2010) Large B cell lymphoma presenting initially in bone marrow, liver and spleen: an aggressive entity associated frequently with haemophagocytic syndrome. Histopathology 57: 785-795. doi:10.1111/j.1365-2559.2010.03709.x. PubMed: 21166693.
[25]
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J et al. (2004) Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 103: 275-282. doi:10.1182/blood-2003-05-1545. PubMed: 14504078.
[26]
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139: 271-279. doi:10.1016/0022-1759(91)90198-O. PubMed: 1710634.
[27]
Chang KC, Chang Y, Jones D, Su IJ (2009) Aberrant expression of cyclin a correlates with morphogenesis of Reed-Sternberg cells in Hodgkin lymphoma. Am J Clin Pathol 132: 50-59. doi:10.1309/AJCPBDFR5L5UOAUZ. PubMed: 19864233.
[28]
Tsou JH, Chang KC, Chang-Liao PY, Yang ST, Lee CT et al. (2011) Aberrantly expressed AURKC enhances the transformation and tumourigenicity of epithelial cells. J Pathol 225: 243-254. doi:10.1002/path.2934. PubMed: 21710690.
[29]
Kaplan EL, Meier P (1958) Nonparametric estimation from incomplete observations. J Am Stat Assoc 53: 457-481. doi:10.1080/01621459.1958.10501452.
[30]
Gehan EA (1965) A generalized two-sample Wilcoxon test for doubly censored data. Biometrika 52: 650-653. doi:10.1093/biomet/52.3-4.650. PubMed: 5858975.
[31]
Bischoff FR, Klebe C, Kretschmer J, Wittinghofer A, Ponstingl H (1994) RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc Natl Acad Sci U S A 91: 2587-2591. doi:10.1073/pnas.91.7.2587. PubMed: 8146159.
[32]
Joseph J, Tan SH, Karpova TS, McNally JG, Dasso M (2002) SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles. J Cell Biol 156: 595-602. doi:10.1083/jcb.200110109. PubMed: 11854305.
[33]
Feng W, Benko AL, Lee JH, Stanford DR, Hopper AK (1999) Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution. J Cell Sci 112 ( 3): 339-347. PubMed: 9885287.
[34]
Katayama H, Brinkley WR, Sen S (2003) The Aurora kinases: role in cell transformation and tumorigenesis. Cancer Metastasis Rev 22: 451-464. doi:10.1023/A:1023789416385. PubMed: 12884918.
[35]
Brunet S, Sardon T, Zimmerman T, Wittmann T, Pepperkok R et al. (2004) Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts. Mol Cell Biol 15: 5318-5328. doi:10.1091/mbc.E04-05-0385. PubMed: 15385625.
[36]
Bai M, Papoudou-Bai A, Kitsoulis P, Horianopoulos N, Kamina S et al. (2005) Cell cycle and apoptosis deregulation in classical Hodgkin lymphomas. In Vivo 19: 439-453. PubMed: 15796209.
[37]
Sánchez-Beato M, Sánchez-Aguilera A, Piris MA (2003) Cell cycle deregulation in B-cell lymphomas. Blood 101: 1220-1235. doi:10.1182/blood-2002-07-2009. PubMed: 12393483.
[38]
Bischoff FR, Krebber H, Kempf T, Hermes I, Ponstingl H (1995) Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. Proc Natl Acad Sci U S A 92: 1749-1753. doi:10.1073/pnas.92.5.1749. PubMed: 7878053.
[39]
Stewart M (2007) Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 8: 195-208. doi:10.1038/nrm2114. PubMed: 17287812.
[40]
Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345-356. doi:10.1016/S0092-8674(02)00630-X. PubMed: 11853669.
[41]
DeGregori J, Russ A, von Melchner H, Rayburn H, Priyaranjan P et al. (1994) A murine homolog of the yeast RNA1 gene is required for postimplantation development. Genes Dev 8: 265-276. doi:10.1101/gad.8.3.265. PubMed: 8314081.
[42]
Rodrigo-Peiris T, Xu XM, Zhao Q, Wang HJ, Meier I (2011) RanGAP is required for post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana. J Exp Bot 62: 2705-2714. doi:10.1093/jxb/erq448. PubMed: 21282324.
[43]
Tsai MY, Zheng Y (2005) Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly. Curr Biol 15: 2156-2163. doi:10.1016/j.cub.2005.10.054. PubMed: 16332542.
[44]
Maresca TJ, Groen AC, Gatlin JC, Ohi R, Mitchison TJ et al. (2009) Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity. Curr Biol 19: 1210-1215. doi:10.1016/j.cub.2009.05.061. PubMed: 19540121.
[45]
Oussenko IA, Holland JF, Reddy EP, Ohnuma T (2011) Effect of ON 01910.Na, an Anticancer Mitotic Inhibitor, on Cell-Cycle Progression Correlates with RanGAP1 Hyperphosphorylation. Cancer Res 71: 4968-4976. doi:10.1158/0008-5472.CAN-10-1603. PubMed: 21646468.
[46]
Pham LV, Zhou HJ, Lin-Lee YC, Tamayo AT, Yoshimura LC et al. (2008) Nuclear tumor necrosis factor receptor-associated factor 6 in lymphoid cells negatively regulates c-Myb-mediated transactivation through small ubiquitin-related modifier-1 modification. J Biol Chem 283: 5081-5089. PubMed: 18093978.
[47]
Chapman CM, Sun X, Roschewski M, Aue G, Farooqui M et al. (2012) ON 01910.Na is selectively cytotoxic for chronic lymphocytic leukemia cells through a dual mechanism of action involving PI3K/AKT inhibition and induction of oxidative stress. Clin Cancer Res 18: 1979-1991.
[48]
Gr？nborg M, Kristiansen TZ, Iwahori A, Chang R, Reddy R et al. (2006) Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach. Mol Cell Proteomics 5: 157-171. PubMed: 16215274.
[49]
Brondyk WH, McKiernan CJ, Fortner KA, Stabila P, Holz RW et al. (1995) Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A. Mol Cell Biol 15: 1137-1143. PubMed: 7532276.
[50]
Kim JA, Kim SJ, Do IG, Jin J, Nam DH et al. (2011) Hypoxia-associated protein expression in primary central nervous system diffuse large B-cell lymphoma: does it predict prognosis? Leuk Lymphoma 52: 205-213. doi:10.3109/10428194.2010.542261. PubMed: 21281236.
[51]
Paulli M, Str？ter J, Gianelli U, Rousset MT, Gambacorta M et al. (1999) Mediastinal B-cell lymphoma: a study of its histomorphologic spectrum based on 109 cases. Hum Pathol 30: 178-187. doi:10.1016/S0046-8177(99)90273-3. PubMed: 10029446.
