Constitutively active nuclear factor- B (NF- B) is integral to the survival of Hodgkin/Reed-Sternberg cells (H/RS) in Hodgkin lymphoma (HL). To investigate NF- B pathway proteins in pediatric HL, we utilized a tissue microarray compiled from 102 children enrolled in the Children's Oncology Group intermediate-risk clinical trial AHOD0031 (56 male, 78 Caucasian, median age 15 years (range 1–20 years), 85 nodular sclerosing subtype, 23 Epstein-Barr virus (EBV) positive, and 24 refractory/relapsed disease). We examined the intensity, localization, and pathway correlations of NF- B pathway proteins (Rel-A/p65, Rel-B, c-Rel, NF- B1, NF- B2, I B- , IKK- , IKK- , IKK- /NEMO, NIK, and A20), as well as their associations with EBV status and clinical outcome. NF- B pathway proteins were overexpressed in pediatric HL patients compared to controls. Patients with EBV？ tumors, or with rapid early therapy response, had tightly coordinated regulation of NF- B pathway proteins, whereas patients with EBV+ tumors, or slow early therapy response, had little coordinated NF- B pathway regulation. High NIK expression was associated with a slow response to therapy and decreased EFS. Elevated Rel-B, NIK, and the NF- B inhibitor A20 were associated with decreased EFS in multivariate analysis. These studies suggest a pivotal role for the NF- B pathway in therapy response and patient survival in Hodgkin lymphoma. 1. Introduction Constitutive activation of NF- B is one of the hallmarks of H/RS cells in HL [1]. NF- B is a transcription factor family composed of five members: Rel-A/p65, Rel-B, c-Rel, NK- B1 (p50/p105), and NF- B2 (p52/p100) [2]. NF- B subunits Rel-A and c-rel are sequestered in the cytoplasm by I B. In the classical pathway (Figure 1(a)), an I B-kinase (IKK) complex regulates I B and the release of Rel-A and c-rel into the nucleus. In the alternative pathway, NF- B is activated by proteasomal cleavage of NF- B2 (p100) to p52, an NF- B subunit that binds and activates Rel-B (Figure 1(b)) [3]. Once activated, NF- B subunits dimerize and translocate into the nucleus, where they activate transcription of NF- B target genes [4–6]. Both the classical and alternative NF- B pathways are tightly regulated. In the classical pathway, degradation of I B- and I B- is triggered by IKK- activation. In the alternative pathway, IKK- is tightly regulated by NF- B inducing kinase (NIK). IKKs are negatively regulated by A20, an E3 ligase/deubiquitinase, and putative tumor suppressor in HL. Figure 1: Classical and alternative NF- B pathway activation. (a) Activation of the classical NF- B
References
[1]
R. Schmitz, J. Stanelle, M. L. Hansmann, and R. Küppers, “Pathogenesis of classical and lymphocyte-predominant hodgkin lymphoma,” Annual Review of Pathology, vol. 4, pp. 151–174, 2009.
[2]
R. Kuppers, “The biology of Hodgkin's lymphoma,” Nature Reviews Cancer, vol. 9, no. 1, pp. 15–27, 2009.
[3]
S. C. Sun and S. C. Ley, “New insights into NF- B regulation and function,” Trends in Immunology, vol. 29, no. 10, pp. 469–478, 2008.
[4]
S. Mathas, A. Lietz, I. Anagnostopoulos et al., “c-FLIP mediates resistance of hodgkin/reed-sternberg cells to death receptor-induced apoptosis,” Journal of Experimental Medicine, vol. 199, no. 8, pp. 1041–1052, 2004.
[5]
H. Kashkar, C. Haefs, H. Shin et al., “XIAP-mediated caspase inhibition in hodgkin's lymphoma-derived B cells,” Journal of Experimental Medicine, vol. 198, no. 2, pp. 341–347, 2003.
[6]
R. K. Thomas, A. Kallenborn, C. Wickenhauser et al., “Constitutive expression of c-FLIP in hodgkin and reed-sternberg cells,” American Journal of Pathology, vol. 160, no. 4, pp. 1521–1528, 2002.
[7]
G. Z. Rassidakis, L. J. Medeiros, T. P. Vassilakopoulos et al., “BCL-2 expression in hodgkin and reed-sternberg cells of classical hodgkin disease predicts a poorer prognosis in patients treated with ABVD or equivalent regimens,” Blood, vol. 100, no. 12, pp. 3935–3941, 2002.
[8]
J. Zhang, H. Quan, J. Ng, and M. E. Stepanavage, “Some statistical methods for multiple endpoints in clinical trials,” Controlled Clinical Trials, vol. 18, no. 3, pp. 204–221, 1997.
[9]
R. C. Bargou, C. Leng, D. Krappmann et al., “High-level nuclear NF-κB and Oct-2 is a common feature of cultured hodgkin/reed-sternberg cells,” Blood, vol. 87, no. 10, pp. 4340–4347, 1996.
[10]
M. Nonaka, R. Horie, K. Itoh, T. Watanabe, N. Yamamoto, and S. Yamaoka, “Aberrant NF-κB2/p52 expression in hodgkin/reed-sternberg cells and CD30-transformed rat fibroblasts,” Oncogene, vol. 24, no. 24, pp. 3976–3986, 2005.
[11]
T. F. E. Barth, J. I. Martin-Subero, S. Joos et al., “Gains of 2p involving the REL locus correlate with nuclear c-Rel protein accumulation in neoplastic cells of classical hodgkin lymphoma,” Blood, vol. 101, no. 9, pp. 3681–3686, 2003.
[12]
S. Mathas, K. J？hrens, S. Joos et al., “Elevated NF-κB p50 complex formation and Bcl-3 expression in classical hodgkin, anaplastic large-cell, and other peripheral T-cell lymphomas,” Blood, vol. 106, no. 13, pp. 4287–4293, 2005.
[13]
K. M. Izumi and E. D. Kieff, “The epstein-barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-κB,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 23, pp. 12592–12597, 1997.
[14]
E. D. C. McFarland, K. M. Izumi, and G. Mosialos, “Epstein-barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-κB,” Oncogene, vol. 18, no. 49, pp. 6959–6964, 1999.
[15]
S. Pai, B. J. O'Sullivan, L. Cooper, R. Thomas, and R. Khanna, “RelB nuclear translocation mediated by C-terminal activator regions of epstein-barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells,” Journal of Virology, vol. 76, no. 4, pp. 1914–1921, 2002.
[16]
I. E. Wartz, K. M. O'Rourke, H. Zhou et al., “De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling,” Nature, vol. 430, no. 7000, pp. 694–699, 2004.
[17]
J. F. Garcia, F. I. Camacho, M. Morente et al., “Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays,” Blood, vol. 101, no. 2, pp. 681–689, 2003.
[18]
C. V. Hedvat, A. Hegde, R. S. K. Chaganti et al., “Application of tissue microarray technology to the study of non-hodgkin's and hodgkin's lymphoma,” Human Pathology, vol. 33, no. 10, pp. 968–974, 2002.
[19]
J. Wang and C. R. Taylor, “Apoptosis and cell cycle-related genes and proteins in classical hodgkin lymphoma: application of tissue microarray technique,” Applied Immunohistochemistry and Molecular Morphology, vol. 11, no. 3, pp. 206–213, 2003.
[20]
A. Sánchez-Aguilera, C. Montalbán, P. De La Cueva et al., “Tumor microenvironment and mitotic checkpoint are key factors in the outcome of classic hodgkin lymphoma,” Blood, vol. 108, no. 2, pp. 662–668, 2006.
[21]
R. C. Bargou, F. Emmerich, D. Krappmann et al., “Constitutive nuclear factor-κb-RelA activation is required for proliferation and survival of hodgkin's disease tumor cells,” Journal of Clinical Investigation, vol. 100, no. 12, pp. 2961–2969, 1997.
[22]
F. Feuerhake, J. L. Kutok, S. Monti et al., “NFκB activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes,” Blood, vol. 106, no. 4, pp. 1392–1399, 2005.
[23]
P. J. Jost and J. Ruland, “Aberrant NF-κB signaling in lymphoma: mechanisms, consequences, and therapeutic implications,” Blood, vol. 109, no. 7, pp. 2700–2707, 2007.
[24]
R. Küppers, “Molecular biology of hodgkin lymphoma,” American Society of Hematology, pp. 491–496, 2009.
[25]
E. Cabannes, G. Khan, F. Aillet, R. F. Jarrett, and R. T. Hay, “Mutations in the IkBa gene in hodgkin's disease suggest a tumour suppressor role for IκBα,” Oncogene, vol. 18, no. 20, pp. 3063–3070, 1999.
[26]
F. Emmerich, M. Meiser, M. Hummel et al., “Overexpression of I kappa B alpha without inhibition of NF-κB activity and mutations in the I kappa B alpha gene in reed-sternberg cells,” Blood, vol. 94, no. 9, pp. 3129–3134, 1999.
[27]
F. Emmerich, S. Theurich, M. Hummel et al., “Inactivating I kappa B epsilon mutations in hodgkin/reed-sternberg cells,” Journal of Pathology, vol. 201, no. 3, pp. 413–420, 2003.
[28]
M. Kato, M. Sanada, I. Kato et al., “Frequent inactivation of A20 in B-cell lymphomas,” Nature, vol. 459, no. 7247, pp. 712–716, 2009.
[29]
R. Schmitz, M. L. Hansmann, V. Bohle et al., “TNFAIP3 (A20) is a tumor suppressor gene in hodgkin lymphoma and primary mediastinal B cell lymphoma,” Journal of Experimental Medicine, vol. 206, no. 5, pp. 981–989, 2009.
[30]
C. Steidl, A. Telenius, S. P. Shah et al., “Genome-wide copy number analysis of hodgkin reed-sternberg cells identifies recurrent imbalances with correlations to treatment outcome,” Blood, vol. 116, no. 3, pp. 418–427, 2010.
[31]
R. Horie, T. Watanabe, Y. Morishita et al., “Ligand-independent signaling by overexpressed CD30 drives NF-κB activation in hodgkin-reed-sternberg cells,” Oncogene, vol. 21, no. 16, pp. 2493–2503, 2002.
[32]
A. Younes, A. Garg, and B. B. Aggarwal, “Nuclear transcription factor-kappaB in Hodgkin's disease,” Leukemia and Lymphoma, vol. 44, no. 6, pp. 929–935, 2003.
[33]
D. Krappmann, F. Emmerich, U. Kordes, E. Scharschmidt, B. D？rken, and C. Scheidereit, “Molecular mechanisms of constitutive NF-κB/Rel activation in hodgkin/reed-sternberg cells,” Oncogene, vol. 18, no. 4, pp. 943–953, 1999.
[34]
E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, “Epstein-barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor,” The EMBO Journal, vol. 17, no. 6, pp. 1700–1709, 1998.
[35]
L. S. Young and P. G. Murray, “Epstein-barr virus and oncogenesis: from latent genes to tumours,” Oncogene, vol. 22, no. 33, pp. 5108–5121, 2003.
[36]
A. G. Eliopoulos, C. W. Dawson, G. Mosialos et al., “CD40-induced growth inhibition in epithelial cells is mimicked by epstein-barr virus-encoded LMP1: involvement of TRAF3 as a common mediator,” Oncogene, vol. 13, no. 10, pp. 2243–2254, 1996.
[37]
F. Schneider, J. Neugebauer, J. Griese et al., “The viral oncoprotein LMP1 exploits TRADD for signaling by masking its apoptotic activity,” PLoS Biology, vol. 6, no. 1, p. e8, 2008.
[38]
G. Khan, “Epstein-barr virus, cytokines, and inflammation: a cocktail for the pathogenesis of hodgkin's lymphoma?” Experimental Hematology, vol. 34, no. 4, pp. 399–406, 2006.
[39]
I. S. Lee, S. H. Kim, H. G. Song, and S. H. Park, “The molecular basis for the generation of hodgkin and reed-sternberg cells in hodgkin's lymphoma,” International Journal of Hematology, vol. 77, no. 4, pp. 330–335, 2003.
[40]
Y. Saitoh, N. Yamamoto, M. Z. Dewan et al., “Overexpressed NF-κB-inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and hodgkin reed-sternberg cells,” Blood, vol. 111, no. 10, pp. 5118–5129, 2008.
[41]
C. Allen, K. Saigal, L. Nottingham, P. Arun, Z. Chen, and C. Van Waes, “Bortezomib-induced apoptosis with limited clinical response is accompanied by inhibition of canonical but not alternative nuclear factor-κB subunits in head and neck cancer,” Clinical Cancer Research, vol. 14, no. 13, pp. 4175–4185, 2008.
