Joseph C. ChengLaboratory of Dr. James S. Norris

Department of Microbiology & ImmunologyMedical University of South Carolina

October 31, 2007

Modulation of Sphingolipid Metabolism Enhances Apoptin’s Cytotoxicity in Prostate

Cancer

VP1 VP3VP2

Chicken Anemia Virus (CAV):

Apoptosis induction

VP1 No

VP2 Weak

VP3 Strong ApoptinApoptin

Noteborn, MH et al. (1994) JVI 68:346-351.

Nur77Nur77

ApoptinApoptin

How Apoptin Works in Cells

Crm1Crm1

ShieldingAggregationUbquitinationDegradation

PP

ApoptinApoptin

Nur77Nur77

ApoptinApoptin

PP

MitochondriaBcl-2Bcl-2

Nur77Nur77

Nur77Nur77

Nur77Nur77

Nur77Nur77

Nur77Nur77 Cytochrome C

Caspase 9

Caspase 3

Apoptosis

DEDAF

APC-1

Hippi

Prostate Cancer

• 217,000 new cases per year in U.S., (670,000 worldwide)

• 27,000 deaths per year in U.S.• 2nd leading cause of cancer

death in American men.• Improved methodologies of

diagnosis and treatment have led to higher cure rate.

• Cancer-related deaths are due to advanced disease by aggressive and resistant cancers.

AdGFPApoptinTET Vector

5' 3'

E1

E3 E4

SV40poly A

tTA

ITR

+ - tetracyclineor doxycycline

TETR +VP16

GFPApoptin TRE ITR

VP16

rTETR

SV40poly A

VP16

rTETR

VP16

rTETR

CMVPromoter

PC

-3

DU

145

LN

CaP

Bcl-2

Bax

FLIPL

FLIPS

DU

145

PC

-3

LN

CaP

Survivin

cIAP-1

XIAP

Bcl-xL

tubulin

Endogenous Gene Expression in Prostate Cancer Cells

DU145 (p53mt/mt), LNCaP (p53wt/wt), and PC-3 (p53null)

Liu et al. (2006) Mol Ther. 14:637-46.

Caspase 3

32KD

17KD

12KD

Ad-GFP Ad-Apop Ad-GFP Ad-Apop Ad-GFP Ad-Apop

DU145 LNcap PC3

Bak

Bax

P-p53

Actin

Apoptin Causes Caspase 3 Dependent Apoptosis in Prostate Cancer Cells

Liu et al. (2006) Mol Ther. 14:637-46.

Prostate Cancer Cell Lines Show Similar Sensitivity to Ad-Apoptin

Liu et al. (2006) Mol Ther. 14:637-46.

CeramideCeramidases

Sphingosine

SphingosineKinase

S1P

Growth inhibition (cell cycle arrest)Apoptosis

Differentiation

Modulation of telomerase activity (telomere length)Senescence

(Pro-apoptotic phenotype)(Pro-apoptotic phenotype)

Cell proliferation

TransformationAngiogenesis

Cell motility (endothelial)

(Anti-apoptotic phenotype)(Anti-apoptotic phenotype)

S1PP

Stress (growth factor withdrawal, hypoxia,

hyperthermia, DNA damage)

Apoptin

Radiation

FasL/AdGFPFasLChemotherapy

0

20

40

60

80

100

120

140

160

180

200

0 10 20 30 40 50 60

Hours post-infection

% C

on

tro

lCeramide

Sphingomyelin

Sphingosine

Apoptin Causes Sphingolipids Changes in DU145 Cells

Liu et al. (2006) Mol Ther. 14:627-36.

Sphingomyelin De novo Synthesis

Ceramide

SMase

Ceramidases

Sphingosine

SphingosineKinase

S1P

Ceramide Synthase

S1PP

Apoptin

The importance of sphingomyelin hydrolysis in apoptosis

• Lymphoblasts derived from patients with acid SMase deficiency (NPD), failed to undergo apoptosis in response to irradiation or CD95 ligation.

• Radiation exposure of thymocytes from acid SMase knockout mice did not undergo apoptosis.

• Ceramide generation induced by addition of exogenous acid SMase augmented apoptosis in human leukemic and prostate cancer cells.

Santana, P. et al. (1996) Cell 86:189.De Maria, R. et al. (1998) J. Exp. Med. 187:897.Monney, L. et al. Eur. J. Biochem. 251:295.Condorelli, F. et al. (1999) Br. J. Pharmacol. 127:75.

RTPCR for Acid Sphingomyelinase (ASMase)/Acid Ceramidase (AC)

Rig/S15

ASMase

Control 6hrs 16hrs 30hrs 48hrs Control 6hrs 16hrs 30hrs 48hrs

Ad-GFP Ad-Apoptin

AC

Western blot

ASMase

Ad-GFP Ad-GFPApoptin

Acid Ceramidase

Actin

Sphingomyelin

Ceramide

Sphingosine

ASMase

Acid Ceramidase

30 hours post-infection

Liu et al. (2006) Mol Ther. 14:627-36.

Translocation of Acid SMase by Confocal Microscopy Detection

16 hours post-infection

Ad-GFP

GFP (Green) ASMase (Red) Overlay

Ad-GFPApoptin

Liu et al. (2006) Mol Ther. 14:627-36.

Ad-Apoptin Increases ASMase Activity

MOI

Liu et al. (2006) Mol Ther. 14:627-36.

Desipramine Partly Delays Apoptin-induced Cell Death

DU145 Co-treated with Ad-Apoptin and Desipramine (1uM and 2.5 uM)

20 30 40 50 60

MOI of Ad-Apoptin

Cel

l V

iab

ilit

y (%

)

0

10

20

30

40

50

60

70

80

90

* p<0.01

* p<0.01

Ad-GFPApoptinApoptin+ Desipramine (1 uM)Apoptin+Desipramine (2.5 uM)

Liu et al. (2006) Mol Ther. 14:627-36.

PKC delta-siRNAScrambled sequence siRNA

Ad-GFP

Ad-GFPApoptin

Ceramide level in PC3 Cells treated with PKC-siRNA

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

C-18:1-Cer C14-Cer C16-Cer C18-Cer C20-Cer C24-Cer C24:1-Cer

XL-1-Scramble

XL-1-pkc-SiRNA

Summary• Tumor-selective viral protein Apoptin induces apoptosis in prostate cancer cells.

• There was no obvious correlation between Apoptin-induced cell death and the status of pro- and anti-apoptotic molecules.

• Apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway.

• Apoptin induces acid sphingomyelinase translocation and activation through PKC.

• Inhibition of acid sphingomyelinase reduces the efficacy of apoptin-induced cell death.

Ceramide

Acid Ceramidase

Sphingosine Sphingosine-1-P

Sphingosine Kinase

Angiogenesis Anti-apoptosis

Ceramide/S1P Pathway

Pro- apoptosis

>60% Gleason grades 5-6 tissues over-express AC.>80% Gleason grades 8-10 tissues over-express AC.

Apoptin

Over-expression of AC Protect Apoptin’s Killing

Cytotoxicity of Ad-Apoptin in DU145 cells Over-expressing Acid Ceramidase

Cel

l Via

bili

ty

0

20

40

60

80

20 40 60 80 100 150

MOI

DU145-EGFP

DU145-ACEGFP#3

DU145-ACEGFP#7

Mock #3 #7

Liu et al. (2006) Mol Ther. 14:637-46.

HN

OH

HO NO2

. HCl

LCL204: Acid Ceramidase Inhibitor

LIPIDOMICS CORE

• AC inhibition by LCL204 results in ceramide accumulation and conversion from an anti-apoptotic phenotype to a pro-apoptotic phenotype.

• LCL204 displays lysomotropic properties by causing rapid lysosomal membane permeabilization (LMP) resulting in translocation of the lysosomal proteases cathepsins B and D into the cytosol.

• Apoptosis induced by LCL204 is dependent on Bak, suggesting that LMP induces a mitochondrial apoptotic pathway.

• LCL204 significantly down-regulates anti-apoptotic genes Flip and Survivin.

Previous Studies:

Holman et al. 2007 Cancer Chemother Pharmacol DOI: 10.1007/s00280-007-0465-0

Acid Ceramidase Inhibitor LCL204 Enhanced Apoptin’s Effect

DU145 Cells Treated with Ad-GFPApoptin (MOI 20)) and Followed by LCL204 (5 uM)

0

20

40

60

80

100

120

NT LCL 204 Ad-GFPApoptin Ad-GFPApoptin+LCL

Treatments

Cel

l Via

bil

ity

(%)

* #

* # ^

Liu et al. (2006) Mol Ther. 14:637-46.

Tumors are treated with 5 intraperitoneal injections of LCL204 75 mg/kg (Q 3 days).

Tumors are treated with 4 intratumoral injections of 2 X 109 PFU adenovirus (Q 3 days).

Design of in vivo experiments

0

100

200

300

400

500

600

700

800

0 5 10 15 20 25 30

Days

Rel

ativ

e tu

mor

vol

um

e

(% o

f or

igin

al)

Control

LCL-204

Apoptin

Apoptin+LCL

*#

*

Animal Study

Liu et al. (2006) Mol Ther. 14:637-46.

Days0 20 40 60 80

Su

rviv

al R

ate

(%)

0

20

40

60

80

100

LCL204

Apoptin

Control

Apoptin+LCL

Animal Study

Liu et al. (2006) Mol Ther. 14:637-46.

Summary

• Apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway.

• Ceramide accumulates in response to Apoptin via increased biosynthesis (ASMase) and retention (AC).

• Pretreatment of prostate cancer cells with AC inhibitor sensitizes tumors to Apoptin, indicating AC is a potential therapeutic target.