FasL: a double-edged sword for tumor formation

Differential effects of FasL on tumor formation Cell-cell contact suppresses Fas-mediated

apoptosis Unconventional Fas signal for gene expression FasL reverse signaling

楊倍昌國立成功大學醫學院微生物及免疫學研究所

BC Yang

Fight cancer, But, sometimes we lose

BC Yang

The great escape: Resistance to killing

• Defective Fas pathway• Resistance to Granzyme

• Fas-L and Neutrophil

Cytotoxic T cells

Innate immunity

BC Yang

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

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Caspase 3 Apaf-1Caspase 9Death substrates

Apoptosis

Nature 407, 789 – 795(2000)

Nagata, S. (Fas); Krammer, P. (Apo1)

CD95/CD95-L

Transducing death signal

Involving in cytotoxicity, immune homeostasis, tumor formation, etc.

BC Yang

In vivo consequences of FasL expression by tumors

(using over-expression system)

Enhanced rejection Delayed rejection

Renca, MH134, L5178Y, B16/BL6, CT26*

Note: *:syngenic, nude, SCID #: allogenic, lpr

Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol.

CT26#

B16-F10

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FasLribozyme/EGFP plasmid

Hammerhead ribozyme.

54-mers. Inserted into the

pEGFP-N1. Driven by CMV

promoter.

Chio CC, Wang YS, Chen YL, Lin SJ and Yang 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

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U-373MG cells transfected withFasLribozyme/EGFP plasmid

Light microscopy

Fluorescent microscopy Immunohistochemistry (tumor cells in tissue)

-tubulinFas-ligand

U37

3(v)

U37

3(R

)U

118(

v)U

118(

R)

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Tumor formation in nude mice

FasLhigh

FasLlow

Br J Cancer (2001) 85, 1185

• Tumor cells

• T, B, Macrophages, monocytes, granulocytes, etc….

• Stroma cells

• Blood vessels

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Q: How about the action of tumor FasL in immune intact mice?

A: It is organ dependent!

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Animal model

C57B/6 Melanoma cell line

B6F10 Gene knockdown by

FasL ribozyme Subcutis/lung

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Effect of FasL on growth and death of B16F10 cells

Apoptosis in 3-day cultureCell growth rate

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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)

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Depletion of CD4, CD8 T cells and PMNs affected lung metastasis

4410

41 357 95154

Vector controls Fas-Lribozyme

What happened here?Br J Cancer, 87: 359 (2002)

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Control

Fas-Lribozyme

In subcutaneous environment: FasL on tumor cells mediates inactivation of neutrophils

5x105cells subcutaneous injection

FasL on tumor cells mediates inactivation of neutrophils. J. Immunol. (2003) 171:1183-1192

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Vector controls Fas-L-ribozyme

Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation

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Tumor FasLhigh impairs neutrophil activation: gelatinase B, ROS, CD-11b, and FasL (In coculture for 24 h).

Cell death

Gelatinase B ROS

J. Immunol. (2003) 171:1183-1192

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Puzzles on tissue specificity (to be answered)

Factors responsible for tissue specificity

Function of immune cells in different tissues

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No death occurred in Jurkat cells during coculture with gliomas

PI-staining

A B

C D E

A: Jurkat cells alone

B: CH-11 (1ng/ml)

C: with U-118MG(V)

D: with U118MG (R)

E: ZB4 (100ng/ml)/CH-11

免疫細胞沒那麼容易死亡 :

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Coculture with tumor cells inhibits the Fas-mediated apoptosis.

Can ECM play a role in this protection?

0.0

10.0

20.0

30.0

40.0

50.0

60.0

J CH-11 U118V U118V+CH-11

U118R U118R+CH-11

HeLa HeLa+CH-11

Huh-7 Huh-7 +CH-11

A549 A549 +CH-11

Apo

ptos

is (%

)

24 h 48 h

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Death pathway inhibited

DISC formation reduced

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

mediumCH-11

U118(V)

U118(V)+CH-11

U118(R)

U118(R)+CH-11

U118(V)+CH-11 (upper)

U118(V)+CH-11 (lower)

U118(R)+CH-11 (upper)

U118(R)+CH-11 (lower)

% A

po

pto

sis

U 118(V/R)

Jurkat cells

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Cell-cell contact is required and integrins involved.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

medium U118(V) CH-11 None blocking anti-β1

blocking anti-β2

blocking anti-β1+β2

% A

nnex

in V

-pos

itive

cel

ls

c

Coculture with glioma cells/conditioned media did not reduced the surface Fas protein.

BC Yang

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

None PD98059 U0126 SP600125 SB202190 GF190203X H-89 LY294002

% A

po

pto

sis

JJ+U118J+CH-11J+U118+CH-11

BC Yang

Intracellular signaling induced by cell-cell contact：PI3K/AKT is important for suppression of Fas-mediated apoptosis in T cells

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Other anti-apoptosis genes

J Immunol. 2007, in revision

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Phosphatidylinositol 3-Kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells

Fas/FasL interaction may create an immunosuppresive environment

• Induction of IL-10 in T cell lines upon contact with human glioma cells, that is mediated by Fas signaling through a PKA-independent pathway.

J Immunol (2003) 171:3947-3954.

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Jurkat Molt-4 1 2 3 1 2 3 IL-10

-actin

Glioma-associated IL-10 induction in T cell lines

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1. control, Jurkat alone2. Jurkat + U118MG3. Jurkat + U373MG

U373(V/R) or U118(V/R)

Jurkat cells 1 2 3 4

IL-10

-actin

Direct cell-to-cell contact is required

1. Jurkat alone2. Jurkat separate chamber3. Jurkat in upper chamber /coculture in low chamber4. Jurkat in coculture

U118(V)

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Fas/Fas-L signal-associated IL-10 induction

Jurkat U-373MG U-118MG - V R V R IL-10 -actin

Molt-4 U-373MG U-118MG - V R V R IL-10

-actin

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Fas

FasL

T cellsTumor

Tissue environment ?

Cytokines ? IL-10, IL-6, IL-8

Neutrophils

To help or not to help ?

To be solved

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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

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3T3-N1 3T3-FasL 3T3-△33 3T3-Fas3T3-△70

FasL on lung metastases

Manuscript in preparation; 2007

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