FasL: a double-edged sword for tumor formation Differential effects of FasL on tumor formation...

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)

BC Yang

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

BC Yang

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

BC Yang

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)

BC Yang

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

BC Yang

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

A: It is organ dependent!

BC Yang

Animal model

C57B/6 Melanoma cell line

B6F10 Gene knockdown by

FasL ribozyme Subcutis/lung

BC Yang

Effect of FasL on growth and death of B16F10 cells

Apoptosis in 3-day cultureCell growth rate

BC Yang

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)

BC Yang

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)

BC Yang

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

BC Yang

Vector controls Fas-L-ribozyme

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

BC Yang

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

BC Yang

Puzzles on tissue specificity (to be answered)

Factors responsible for tissue specificity

Function of immune cells in different tissues

BC Yang

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 :

BC Yang

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

BC Yang

BC Yang

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

BC Yang

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

BC Yang

Other anti-apoptosis genes

J Immunol. 2007, in revision

BC Yang

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.

BC Yang

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

-actin

Glioma-associated IL-10 induction in T cell lines

BC Yang

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)

BC Yang

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

BC Yang

Fas

FasL

T cellsTumor

Tissue environment ?

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

Neutrophils

To help or not to help ?

To be solved

BC Yang

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

BC Yang

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

FasL on lung metastases

Manuscript in preparation; 2007

BC Yang