1 Fas Ligand: switching signals from tumor immune erosion to metastasis Bei-Chang Yang, Ph.D. ( 楊倍昌 ) Professor, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University (NCKU), Tainan, Taiwan. Director, Research Center for Society, Technology and Medicine (STM center); NCKU Tainan, Taiwan.

2 On Tumor microenvironment and infiltrating T cells Novel FasL signaling mechanism!  (FasL) Lin HC, et al (2013) J Biol Chem. 287:20664– Why are T cells trapped in peripheral area of tumor?  (Tenascin C) Huang JY, et al (2010) J Immunol. 185:  (collagen) Lin YP, et al. (2009) Mol Immunol. Mol Immunol. 46: Are those infiltrating T cells alive ?  (PI3K) Su CC, et al (2007) J Immunol. 179:  (FasL) Hor WS, et al. (2003) J Leukocyte Biol. 73: Do infiltrating T cells affect tumor growth or metastasis?  (depletion study) Chen YL, et al (2002) Br J Cancer 87:  (Fas signaling) Chen YL, et al. (2003) J Immunol. 171: Can they modulate consequent local immune reaction?  (IL-10 & TGF-b) Yang BC, et al. (2003) J Immunol. 171:  (Fas and Th17) Su CC, et al (2013) Cell Immunol. 281:101–110 FasL+ Fas+

3 Fas/Fas-ligand : the major signaling system for cell death. Fas (1991): Nagata, S.; Krammer, P. FasL (1993): Nagata, S. & Goldstein, P. CD95/CD95-L Fas: Itoh N, et al. (1991) Cell 66: 233–243 (Nagata S) Oehm A, et al. (1992) J. Biol. Chem. 267: 10709–10715 (Krammer P) FasL: Suda T, et al. (1993) Cell 75: (Nagata S; Goldstein P) 長田 重一 P Krammer apoptosis

4 Caspase 3 Apaf-1 Caspase 9 Death substrates Nature 407, 789 – 795(2000)

5 FasL reverse signaling (FasL by itself acts as a receptor) Suzuki I. and Fink PJ, Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand. J. Exp. Med., 187:123, Sun M, Ames KT, Suzuki I,. Fink PJ, The Cytoplasmic Domain of Fas Ligand Costimulates TCR Signals, J. Immunol., 177, 1481, BC Yang Pamela J. Fink, h.D. Department of Immunology University of Washington The cytoplasmic domain of FasL is sufficient to mediate costimulation (FasL cross-linking is required).

6 Conventional view of receptor-ligand interaction FasL Fas FasL

7 Protein-protein interaction in lipid raft is an alternative way for cell signaling. Take home massage: Fas ligand hijack the Met receptor on lipid raft.

8 The structure of FasL

9 (A). Full-length and truncated FasL. Adapted from Orlinick JR, et al. J Biol Chem 1997, 272: Jodo S, et al. J Immunol 2005, 174: FasL- △ 70: deleting the N-terminal 2-70 aa; FasL- △ 33: deleting the N-terminal 2-33 aa. Due to unstable protein region

10 (A) 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. (B) 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) FasL enhances cell migration and invasion. A B

11 anchorage-independent growth. Truncated FasL promote cell transformation (enhances lung metastasis, anchorage-indecent growth) Lung metastasis in eight-week-old BALB/cAnN.Cg-Foxn1nu/CrlNarl nude mice

12 The Met tyrosine kinase receptor is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF-Met system is necessary for embryonic development, and aberrant MET signaling favors tumorigenesis and metastasis. cyclinD1 Metastasis Q: Why FasL without intracellular domain still promotes tumor metastasis?

13 Association of FasL and Met in lipid raft Location of Met is not changed Lipid raft formation is required M  CD:methyl-beta-cyclodextrin, a specific cholesterol-binding agent extracting cholesterol from plasma membranes

14 Confocal imaging reveals colocalization of FasL and Met MF/M: Met/FasL complex/total Met FL/L:FasL in lipid raft/lipid raft ML/L:Met in lipid raft/lipid raft labeled cholera toxin B

A FasL domain between is important for complex with Met. MRC5 cells/  FasL (N-20, Senta Cruz) NIH3T3/  FasL (C-terminal, BD) Note: full length FasL triggered profound apoptosis in MRC5 FasL

16 The Met tyrosine kinase receptor is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF-Met system is necessary for embryonic development, and aberrant MET signaling favors tumorigenesis and metastasis. cyclinD1 Metastasis

17 Met signal is initiated and mediates transformed phenotype. Stat3 is essential for the enhanced cell migration. Met inhibitor: PHA Stst3 inhibitor: AG490

18 The complex of FasL and Met is detected in various tumor cell lines. Suppression of FasL by specific ribozyme reduces the phosphorylation of Met. A B FasL/Met complex in human tumor cells

19 Fas/FasL signaling revised: There are three different pathways initiated by FasL. lipid raft Fas counter-attack mechanism/ Tissue destruction? FasL-associated metastasisAutoimmunity?

20 Mother nature works in a way that is not always as been told.