New insight in death signaling: on Fas and FasL

1. New insight in death signaling:on Fas and FasL • Bei-Chang Yang, Ph.D. (楊倍昌) • Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University (NCKU), Tainan, Taiwan. • Research center for Society, Technology and Medicine (STM center); NCKU Tainan, Taiwan.

2. The term 'apoptosis' describes the molecular and morphological processes leading to controlled cellular self-destruction and was first introduced in a publication by Kerr, Wyllie and Currie. Br. J. Cancer, 1972, 26: 239

3. Apoptosis 花瓣樹葉的凋零飄落 'Apoptosis' (pronounced as əpo΄tosis ) is of Greek origin, having the meaning "falling off or dropping off", in analogy to leaves falling from trees or petals from flowers, sometimes also called as programmed cell death. 東豐路, 台南

4. Some types of death: apoptosis, autophage, and necrosis AUTOPHAGE

5. Detection of apoptotic cells • Hoechst stain • DNA ladder • Comet assay • TUNEL stain; immunohistochmical (terminal transferase) • FACS analysis (Annexin V, MC540, propidium iodine for subG0, TUNEL)

6. TNF receptor (1985) • Kull FC Jr.  Jacobs S.  Cuatrecasas P.(1985) Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity. PNAS 82:5756-5760.  • Fas (1989) • P Krammer group (1989): Trauth BC.  et al.  Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science  245(4915):301-305. • S Nagata group (1991):   Itoh N.  et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233-243.

8. Shigekazu Nagata Peter H. Krammer, 2002 For a man who spends most of his time thinking about death, Shigekazu Nagata is remarkably upbeat. For over a decade, he has been making a name for himself with research on apoptosis, the mechanism of programmed cell death, and during that time he has watched the field come alive. Nature Medicine  7, 759 (2001)

9. Nature 407, 789 – 795(2000) Apaf-1 Caspase 9 Caspase 3 Death substrates Apoptosis • Nagata, S. (Fas); Krammer, P. (Apo1) • CD95/CD95-L • Transducing death signal • Involving in cytotoxicity, immune homeostasis, tumor formation, etc.

10. Fas counterattack mechanism • Hahne, M., D. Rimoldi, M. Schroter, P. Romero, M. Schreier, L. E. French, P. Schneider, T. Bornand, A. Fontana, D. Lienard, J. C. Cerottini, and J. Tschopp. 1996. Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science 274:1363. • Strand, S., W. J. Hofmann, H. Hung, M. Muller, G. Otto, D. Strand, S. M. Martani, W. Stremmel, P. H. Krammer, and P. R. Galle. 1996. Lymphocyte apoptosis induced by CD95(APO-1/Fas) ligand-expressing tumor cells-a mechanism of immune evasion ? Nature Med.2:1361. • O’Connell, J., G. C. O’Sullivan, J. K. Collins, and F. Shanahan. 1996. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J. Exp. Med.184:1075. • Seino, K., N. Kayagaki, K. Okumura, and H. Yagita. 1997. Antitumor effect of locally produced CD95 ligand. Nature Med. 3:165. • O’Connell, J., M. W. Bennett, G. G. O’sullivan, D. Roche, J. Kelly, J. K. Collins, and F. Shanahan. 1999. Fas counter-attack the best form of tumor defense? Nature Med.5:267. • Seino, K. I., N. Kayagaki, N. Tsukada, K. Fukao, H. Yagita, and K. Okumura. 1997. Transplantation of CD95 ligand-expressing grafts. Influence of transplanation site and difficulty in protecting allo- and xenigrafts. Transplantation64:1050.

11. Many tumor cells express FasL Nat Med  5:267 (1999); Nat Med 7:271 (2001) Joe O'Connell, et al. Fas counter-attack- the best form of tumor defense? Tumor

12. Immune privilege or inflammation?Joe O'Connell et al. Nature Medicine 7,271-274 (2001)

13. Enhanced rejection Delayed rejection In vivo consequences of FasL expression by tumors(using over-expression system) Renca, MH134, L5178Y, B16/BL6, CT26* CT26# B16-F10 Note: *:syngenic, nude, SCID #: allogenic, lpr Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol.

14. Animal model • C57B/6 • Melanoma cell line B6F10 • Gene knockdown by FasL ribozyme • Subcutis/lung

15. FasLribozyme/EGFP plasmid • Hammerhead ribozyme. • 54-mers. • Inserted into the pEGFP-N1. • Driven by CMV promoter. Chio CC, Wang YS, Chen YL, Lin SJ andYang BC (2001) Down-regulation of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumor infiltrating cells, and liver damage but accelerates tumor formation in nude mice. Br J Cancer 85:1185-1192

16. Effect of FasL on growth and death of B16F10 cells Apoptosis in 3-day culture Cell growth rate

17. Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behavior (B16F10 in C57BL/6) Br J Cancer, 87: 359 (2002)

18. Depletion of CD4, CD8 T cells and PMNs affected lung metastasis 44 41 357 95 10 154 Vector controls Fas-Lribozyme What happened here? Br J Cancer, 87: 359 (2002)

19. In subcutaneous environment: FasL on tumor cells mediates inactivation of neutrophils 5x105cells subcutaneous injection Control Fas-Lribozyme FasL on tumor cells mediates inactivation of neutrophils. J. Immunol. (2003) 171:1183-1192

20. Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation Vector controls Fas-L-ribozyme

21. Cell death • Tumor FasLhigh impairs neutrophil activation: gelatinase B, ROS, CD-11b, and FasL (In coculture for 24 h). Gelatinase B ROS J. Immunol. (2003) 171:1183-1192

22. The action of FasL: It is organ dependent!

23. Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behavior (B16F10 in C57BL/6) Br J Cancer, 87: 359 (2002) i.v. 5 x 105 cells /mouse; Observed at day 14; (bar = 1 cm) • Voice against Fas counter attack mechanism: • Kang, S. M. et al. 1997. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nature Med.3:738.

24. Ryan AE,1 Shanahan F, O’Connell J, and Houston AM (2005) Addressing the ‘‘Fas Counterattack’’ Controversy: Blocking Fas Ligand Expression Suppresses Tumor Immune Evasion of Colon Cancer In vivo. Cancer Res 65: 9817-9823 (困獸之鬥?) • Figure 1. FasL antisense RNA suppresses FasL expression by CMT93 cells. • Figure 2. Effect of reduced FasL expression by CMT93 cells on tumor development. • Figure 3. Analysis of tumors derived from CMT93/AS, CMT93/C and parental cells inoculated s.c. in C57BL/6 mice. • Figure 4. Down-regulation of FasL expression leads to increased infiltration of TILs. • Figure 5. Constitutively expressed FasL does not trigger neutrophil recruitment in vivo. No Fas-mediated apoptosis at all! What Joe O’Connell was thinking then? Increase in Th17 cells

25. FasL reverse signaling • Suzuki I. and Fink PJ.. Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand. J. Exp. Med., 187:123, 1998. • Boursalian TE, Fink PJ, Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors, J. Exp. Med., 198, 349, 2003. • Sun M, Ames KT, Suzuki I,. Fink PJ, The Cytoplasmic Domain of Fas Ligand Costimulates TCR Signals, J. Immunol., 177, 1481, 2006. Pamela J. Fink, h.D. Department of Immunology University of Washington

26. The structure of FasL

27. The cytoplasmic domain of FasL is sufficient to mediate costimulation. A, Murine FasL and z (CD3)fusion constructs encode an N-terminal myristylation motif (M) that tethers the molecules to the membrane, the relevant cytoplasmic domains, FKBP3, and a C-terminal HA epitope. The addition of FK1012 or AP1510 induces hetero-oligomers of these membrane-bound proteins. B, Jurkat cells cotransfected with combinations of CID constructs, firefly luciferase reporter (NFAT or AP-1) and Renilla luciferase reporter (pRL-TK) were subjected to CID analysis. J. Immunol., 177, 1481, 2006

28. Ectopic expression of FasL in NIH3T3 (A). Full-length and truncated FasL. Adapted from Orlinick JR, et al. J Biol Chem 1997, 272:32221-29. Jodo S, et al. J Immunol 2005, 174:4470-4474. FasL-△70: deleting the N-terminal 2-70 aa; FasL-△33: deleting the N-terminal 2-33 aa.

29. Full-length FasL suppresses lung metastasis, while truncated FasL promotes tumor formation. Nude mice were injected with 5x105 cells of NIH3T3 cells carrying FasL as indicated via tail vein. After 4 weeks, spontaneous transformed NIH3T3 tumor nodules formed in the lung. Ling sections were stained with H/E. Tumor nodules show dark purple stain.

30. Truncated FasL promote cell transformation (enhances anchorage-indecent growth) FasL△33 and FasL△70 enhanced anchorage-independent growth. 1.5 ×104 cells were cultured in 0.3% noble agar for 2-3 weeks. Foci were observed under a microscope. Representative shape of colony (x100) was shown (A). Number of foci was counted (B). Values was shown the mean ± SEM. (* p<0.05 compared with N1).

31. FasL enhances cell migration and invasion. In the single cell motility assay, cell migration distance in 6 h was recorded every 5 min by time lapse video under a microscope. Total distance of cell migration was taken to represent cell mobility. (C) In invasion assay, cells were seeded on matrigel (1 mg/ml) coated transwell plates in 16 h. Invading cells were calculated for quantification. Values was shown the mean ± SEM. (* p<0.05, ** p<0.001 compared with N1, # p<0.05 compared with FasL)

32. Phospho-STAT3 and STAT3 were analyzed by Western blot (B). Cells were incubated in 1% FBS in 16 h and detected the expressions of cyclin D1 by Western blotting (C) and RT-PCR (D).

33. FasL reverse signal, A speculation FasL-△70 Full-length FasL Cell membrane ? PRD ERK, PI3K CK1 Stat3, cyclinD1 Inhibitory signal regulating immune responses Tumor metastasis

37. A proposed FasL-Met signal pathway.

38. Do you still believe in Fas counter- attack mechanism?

39. Nature is working in a way that is not always as been told.