Epstein-Barr virus ( EBV ) and pathogenesis of Nasopharyngeal carcinoma ( NPC)

1. Epstein-Barr virus(EBV) and pathogenesis of Nasopharyngeal carcinoma (NPC)

2. Epstein-Barr virus(EBV) • A human herpesvirus , -subfamily of herpesviridae • 1958- first description of Burkitt's lymphoma • 1964- Epstein and Barr isolate continuous cell lines from • Burkitt lymphoma cells • 1968- EBV demonstrated as the etiological agent of • infectious mononucleosis • 1969- EBV shown to immortalize lymphocytes in culture

3. 172 kb viral genome

5. Epstein-Barr virus(EBV) • Latent Infection • latency I : Epstein-Barr virus nuclear antigens (EBNA1) • latency II: EBNA1 and LMP1, LMP2 • latency III: EBNA1, 2, 3, LP and LMP1, 2 • Lytic Activation • Associated several malignancies • Burkitt's lymphoma, Hodgkin’s Disease, post-transplant • lymphoproliferative disorders,T cell lymphoma • Gastric cancer,Nasopharyngeal carcinoma (NPC)

6. Nasopharyngeal carcinoma (NPC) • Occurs world wide, rare in western countries, high in • south-east Asia, northern Africa, Greenland, and Alaska • Two main histological types of NPC by WHO classification: • (I) Squamous cell carcinoma --- desmoplastic, without lymphoid stroma • (II) Non-keratinizing carcinoma --- associated with lymphoid stroma • differentiated non-keratinizing/undifferentiated carcinoma • (Nicholls, 1996) • Undifferentiated carcinoma associated with prominent lymphoid • stroma ---- the most common form of NPC (~80% of cases)

7. Association of EBV with NPC • Initially suggested by serological studies • Subsequently substantiated by the detection of viral genomes • and gene products in the NPC tumor cells • (Wolf et al., Nature, 1973; Klein et al., PNAS USA, 1974) • Which types of NPC are associated with EBV? The viral genomes is of monoclonal origin in undifferentiated NPC, so virus infection must have occurred before clonal expansion of tumor cell

8. By contract, conflicting results have been reported about association of EBV with squamous cell NPC Squamous cell NPCs from areas where NPC is endemic may be mostly EBV-associated whilst morphologically identical cases from areas where NPC occurs as a sporadic tumor are commonly not EBV-related

9. EBV genomes in epithelial cells of in-situ NPC lesions

10. The complement receptors (CR2/CD21) are the EBV receptors on B cells The regulation of B cell activation by complement receptor Binding of complement proteins (C3d) to antigens directly or via attached antibodies leads to coligation of antigen receptors (Ig) and complement receptors (CR2, CD21) on antigen-specific B cells. This enhances the Ig-mediated signal and the subsequent B cell response. The EBV viral envelope protein gp350/200, which shares a region of sequence similarity with C3d, can interact with CR2/CD21

11. The complement receptors (CR2/CD21) on epithelial cells JOURNAL OF VIROLOGY, 73, 2115–2125 (1999)

12. CD21 Northern blotting RT-PCR for EBNA1 For EBER1

13. 1969- EBV shown to immortalize lymphocytes in culture Latent Infection of EBV --- latency I : Epstein-Barr virus nuclear antigens (EBNA1) latency II: EBNA1 and LMP1, LMP2 latency III: EBNA1, 2, 3, LP and LMP1, 2 What gene product(s) of EBV is (are) responsible for cell transformation?

14. -- B95-8 strain -- -- NPC strain, have 10 amino acid deletion essential in tumorigenesis Chen et al., (1992) Oncogene; Lee et al.,(1996) Oncogene BLMP1 NLMP1 Latent membrane protein (LMP1) • latent membrane proteins: LMP1 and 2A, 2B • LMP1--- present in > 60% of all NPC and Hodgkin's lymphoma • --- can transform rodent fibroblasts, which grow as tumors • in nude mice Wang et al., (1985) Cell; Sugden, B. (1988) Oncogene • --- induce B lyphoma in transgenic mice Raab-Traub, N. (1998) PNAS USA

15. LMP1 structure Hammerschmidt W. (1999) EMBO J. • 62 kDa polypeptide: 25 amino acid at N-terminus • 6 transmembrane domain • 200 amino acid at C-terminus • CTAR: C-terminal activation region • C-terminal required for the signal transduction

16. LMP1 affects multiple signaling events in cells CTAR: C-terminal activation region Hammerschmidt W. (1999) EMBO J. LMP1 acts like a permanently activated receptor

17. Morphological transformation of human keratinocytes expressing the LMP1 gene of Epstein-Barr virus Klein et al. (1990) Nature RHEK-1: non-tumorigenic keratinocyte cell line LMP1 C C LMP1 C LMP1 W.B: AE1/AE3 (cytokeratin 40-66K) W.B: PKK-2 (cytokeratin 19, 17, 16, 7) W.B: PKK-1 (cytokeratin 19, 18, 8) LMP1 transfected RHEK-1 cells shows a down-regulation of cytokeratin expression

18. Control cell Transfected with LMP1 Change in morphology and growth in spindle-shaped bundles LMP expression may be an important causal factor in the development of NPC

19. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cell from programmed cell death Henderson et al. (1991) Cell W.B with LMP1 mAb apoptosis assay EBV-negative BJAB lymphoma cell line LMP1 mediates the protection from apoptosis by up-regulating expression of bcl-2

20. The Epstein-Barr virus LMP induces A20 zinc finger protein expression by activating nuclear factor-B Laherty et al. (1992) J. Biol. Chem. Involvement of two NF-kB binding elements in tumor necrosis factor alpha-, CD40-, and Epstein-Barr virus latent membrane protein 1-mediated induction of the cellular inhibitor of apoptosis protein 2 gene Hong et al. (2000) J. Biol. Chem. A20 and cIAP2 are antiapototic proteins

21. Cellular proteins interacted with LMP1 The Epstein-Barr virus transforming protein LMP-1 engages signaling proteins for the tumor necrosis factor receptor family Mosialos et al. Cell (1995) • The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for B lymphocyte growth transformation and is now shown to interact with a novel human protein (LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein, tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in growth signaling from the p80 TNFR. A second novel protein (EBI6), induced by EBV infection, is the human homolog of a second murine TNFR-associated protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1 clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates with these proteins. LAP1 binds to the p80 TNFR, CD40, and the lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The interaction of LMP1 with these TNFR family-associated proteins is further evidence for their role in signaling and links LMP1-mediated transformation to signal transduction from the TNFR family. • (identified by yeast two-hybrid system)

22. TRAF2 (TNFR-associated factor 2) --- An important signal transducer for TNFR to activate NF-kB pathway

23. 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-kB IZUMI AND KIEFF* PNAS USA (1997) The Epstein–Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model thatLMP1is a constitutively activated receptor that transduces signals for transformation through its carboxylterminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacte with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucinemutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kB activation also appears to be a critical component of long-term outgrowth.

24. TRADD (TNFR-associated death domain protein) --- An important signal transducer for TNFR to activate NF-kB and JNK pathway

25. The Epstein–Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation (NF-kB/TRAF) IZUMI et al., (1997) PNAS USA Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185–211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1D185–211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1D185–211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.

26. LMP1 activates transcription factor NF-B through a pathway that includes the NF- B-inducing kinase and the I B kinases IKK  and IKK  Mosialos et al. PNAS USA (1998) NIK  IKK/  NF-B IKK IKK Dominant negative mutant NIK

27. Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade Hammerschmidt et al. (1997)EMBO J JNK1 is induced by LMP-1 AP-1 induction by LMP-1

28. Dominant-negative mutants of SEK1 block LMP-1-induced activities of JNK1 and AP-1 LMP-1 induces AP-1-dependent transcription via the SEK1  JNK1  c-Jun N-terminus cascade

29. deletion of CTAR2 completely abolished LMP-1’s ability to induce AP-1 The LMP-1 C-terminal CTAR2 region is essential for LMP-1's effects on AP-1

30. LMP1 structure and signal transduction Eliopoulos and Young, Seminars Cancer Biol. (2001) The oncogenic Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has structural features and functions reminiscent of a constitutively active TNF family receptor. LMP1 aggregates at the plasma membrane and initiates the activation of signalling pathways, such as NF-kB, the mitogen-activated protein kinases JNK and p38, the small GTPase Cdc42 and the JAK/STAT cascade. The constitutive engagement of these signals and the characteristic molecular interactions that regulate them provide the basis for the molecular explanation of the transforming properties of this key EBV protein.

31. Rel/NF-kappaB/IkappaB proteins and cancerGilmore et al., Oncogene (1996)

32. The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases Y.S. Chang PNAS USA. (2002) LMP1 Represses E-cadherin Gene Expression CTAR-2 is essential for LMP1-Mediated Down-Regulation of the E-cadherin Gene

33. Two tumor supressor genes are hypermethylated in NPC High Frequency of Promoter Hypermethylation of RASSF1A in Nasopharyngeal Carcinoma Lo et al., Cancer Res (2001) Hypermethylation of the p16 gene in nasopharyngeal carcinoma Lo et al., Cancer Res (1996)

34. yeast two-hybrid assay LMP1 C-T BRAM1 Bait: LMP1cytoplasmic domain cDNA library:BALB/c3T3 cells Negative Regulation of Epstein-Barr Virus Latent Membrane Protein 1-mediated functions by Bone Morphogenetic Protein Receptoe IA-Binding Protein, BRAM1 Chung et al., J. Biol. Chem., October 18, 2002 Full-length BRAM1 (WT) and its deletion mutants

35. Identification of the interacting domains of BRAM1 and LMP1 Complete C-terminal CTAR2 and intervening CTAR1 CTAR1 and intervening C.T excluding YYD The C-terminal 116-amino acid sequence of BRAM1 specifically interacts with CTAR2 of LMP1

36. BRAM1---Bone morphogenetic protein receptor IA-binding protein • TGF- superfamily: Ligand family Receptor family TGF- Receptor BMP Receptor TGF-  BMP Nodal • Biological fuction: Regulate pattern formation (during development) Influence cell behaviors: proliferation; cell death differentiation extracellular matrix production Tissue repair Immune regulation

37. Cellular localization of LMP1 and BRAM1 conventional fluorescence microscope NPC-TW02 cells  transfected with GFP-LMP1 and Flag-BRAM1  anti-Flag immunofluorescence staining  observed with luciferase microscope confocal microscope in XY scanning confocal microscope in XZ scanning BRAM1 and LMP1 are co-localized in cells

38. LMP1 ? BRAM1 293 cells  transfected with B/N-LMP1 BRAM1  in vitro kinase assay BALB/c3T3 cells  transfected with 4xB-Luc B/N-LMP1, Flag-BRAM1  luciferase values  WB using anti-LMP1 (S12) and anti-Flag (M2 ) BRAM1

39. Induction of inducible nitric oxide synthase by Epstein-Barr virus B95-8-derived LMP1 in Balb/3T3 cells promotes stress-induced cell death and impairs LMP1-mediated transformation Yu et al., Oncogene in press (2002) Balb/3T3 cells BLMP1(+++) NLMP1 (+) iNOS induction iNOS inhibitor NO production More sensitive to stress (UV/HS) Reduced anchorage-independent growth Reduced tumorigenecity in nude mice (?) HS

40. Effects of iNOS induction on the tumorigenecity of LMP1-expressing Balb/3T3 cells in nude mice model E9 D11 D13 -AG +AG

41. TRAF2 TRADD Multiple signaling cascades (NF-kB, AP-1) Pleiotropic effects on cell functions (A20, cIAPs, IL-6, IL-8, CD40, ICAM1, transformation) Molecules That Interfere Interactions between LMP1/TRADD (or TRAF2) May Represent Lead Compounds for Potential Anti-EBV Drug Development

42. ELISA for Measuring the Interactions between EBV LMP1 and Its Downstream Signaling Molecules: TRAF2 and TRADD

43. ELISA for Interactions between LMP1 and TRAF2/TRADD

44. ELISA for Interactions between biotin-labeled LMP1C16 peptide and TRADD

45. Thank you for your attention

46. TNFR LMP1 ? BRAM1 BRAM1

47. Fig. 4.Whether BRAM1 is also involved in TNF signaling 293 cells  transfected with Flag-BRAM1  treatment with TNF-  luciferase activity BRAM1 acts as a negative regulator for NF-B activation in response to TNF- treatment

48. TNFR LMP1 ? ? BRAM1 BRAM1 BRAM1 BRAM1

49. Fig. 5A. Whether BRAM1 abrogate NF-κB activation through preventing the phosphorylation and degradation of IκBα BALB/c3T3 cells  transfected with B-LMP1 and/or BRAM1  WB using anti-phospho-IB antibody (B-9) and anti-IB-specific antibody (C-21) BRAM1 prevented LMP1-induced IκBα phosphorylation and degradation

50. Fig. 5B.C. The role of BRAM1in the inhibition of phosphorylation and degradation of IB BALB/c3T3 cells  transfected with BRAM1  treated with TNF-  WB using anti-IB antibody (C-21) and anti-phospho-IB antibody (B-9) BRAM1 significantly reduced the IκBα activation in response to TNF-α treatment