Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans

TUMOR CELL-SPECIFIC NUCLEAR TARGETING BY

CAV VP3 (APOPTIN): PROMISES FOR ANTI- CANCER THERAPY ?

Nuclear Signalling Lab.

Dept. Biochem. & Mol. Biol.Monash University

(Melbourne, Australia)

Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff

and David A. Jans

Anti-Cancer Approaches

– Cancer is a growing problem world-wide

– We have drugs that kill tumor (but also normal) cells

efficiently (ultimately the patient)

– There is an urgent need to be able to deliver drugs

specifically to tumor cells, and yet retain efficiency in

anti-cancer therapy

Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50

Viral/non-viral mechanisms of drug delivery: Cellular barriers to bioactive

molecules

Anti-Cancer Approaches– Cancer is a growing problem world-wide

– We have drugs that kill tumor (but also normal) cells

– There is an urgent need to be able to deliver drugs specifically to tumor cells, and

yet retain efficiency in anti-cancer therapy

– Cancer cells derive from normal cells; the idea of a magic bullet to

combat cancer (a single property of a cancer cell that can be targeted

specifically) may be naïve (Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50)

– VP3/apoptin – a viral gene-product with tumor cell-specific activity

– ssDNA virus

– Causes immunosuppression /severe

anaemia in young chicks by inducing

apoptosis in thymocytes/ erythroid

progenitors

– 3 viral proteins (VPs): VP1, VP2 and VP3

Chicken Anemia Virus (circovirus)

http://www.tanglewoodfarms.net/img/poulet-rouge/Cou-nus-young1.jpg

http://www.tanglewoodfarms.net/img/poulet-rouge/Cou-nus-young1.jpg

VP3/Apoptin

VP3

Nucleus

Cytoplasm

Tumor/transformed cells Normal/untransformed cells

• Nuclear localization

• Induces apoptosis

• Cytoplasmic localization

• No apoptosis

Non-isogenic cell comparisons (eg. SAOS-2 human osteosarcoma line v. VH10 foreskin

fibroblasts Transformed human embryonic kidney v. Rat primary

hepatocytes)

CYTOPLASM NUCLEUS

NLSNLS

Importin 1-Mediated Nuclear Import

NESNES

NLS

NUCLEARENVELOPE

NPC

MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH

THE NPC


IMPx (exportin)-Mediated Nuclear Export

c. 12 distinct EXPs

RanGTP

RanGTP

IMP1

IMP IMP

Exportin

Exportin

NLS

NLS, nuclear localisation

signal (generally

Lys/Arg-rich)

Nuclear Pore Complex(nups – nucleoporins)

RanGTP

RanGTP

CYTOPLASM NUCLEUS

NLSNLS

NLS

NUCLEARENVELOPE

NPC


THE NPC

IMP1

IMP IMP

NLS


signal (generally

Lys/Arg-rich)

RanGTP

RanGTP

NESRanGTP

Exportin

NES

Exportin Exportin 1 (CRM1)-

Mediated Nuclear Export

NES, nuclear export signal

(generally hydrophobic) Ran

GDP



CYTOPLASM NUCLEUS

NLSNLS

NLS

NUCLEARENVELOPE

NPC


THE NPC

IMP1

IMP IMP

NLS


signal (generally

Lys/Arg-rich)

RanGTP

RanGTP

NESRanGTP

Exportin

NES

Exportin Exportin 1 (CRM1)-

Mediated Nuclear Export

RanGDP



X X

LMB, leptomycin B:

CRM1-specific inhibitor

VP3/Apoptin

VP3

Nucleus

Cytoplasm





• No apoptosis

Nuclear Targeting Signals in VP3

IRIGIAGITITLSL

LRS NLS1 NES NLS2

1 33 46 82 88 97 105 111 121

KPPSKKR SCDPSEYR VSKLKESLI TT T108 PS RPRTAKRRIKL

N C

NLS: Nuclear Localization Sequence

NES: Nuclear Export Sequence

Is VP3 recognised by Importins or Exportins ?

AlphaScreen Binding Assay

S bIMPNi2

Acceptor

beads

Donor

beads

.O2 Emission:

620nm

Ni2Donor

beads

.O2

S Acceptor beads

Binding

No binding

VP3-H6

VP3-H6

No Emission

Excitation:

680nm

bImps: biotinylated Imps

VP3-His6: hexa-His-tagged GFP-VP3-HMG

S: streptavidin

b: biotin

680nm

(Amplified Luminescence Proximity Homogeneous Assay)

bIMP

200 nm

Wagstaff and Jans (2005) Anal. Biochem. 348, 49-56

GFP-VP3(1-121)

IMP (nM)

0 10 20 30 40 50 60

% M

axim

al B

ind

ing

0

20

40

60

80

100+ IMP

Kd = 9.7 nM

+ IMP

+ IMP


VP3 is recognised with high affinity by IMP1; modulated by intramolecular

masking

GFP1 33 46 82 88 97 105 111 121

LRS NLS1 NES NLS2 1-121

GFP-VP3(74-121)

IMP (nM)

0 10 20 30 40 50 600

20

40

60

80

100+ IMP

Kd = 3.7 nM

+ IMP

+ IMP

74-121GFP NLS1 NES NLS2

Nuclear Targeting Signals in VP3

IRIGIAGITITLSL

LRS NLS1 NES NLS2

1 33 46 82 88 97 105 111 121

KPPSKKR SCDPSEYR VSKLKESLI TT T108 PS RPRTAKRRIKL

N C

NLS: Nuclear Localization Sequence

NES: Nuclear Export Sequence

Thr108: phosphorylated specifically in tumor cells

Rohn et al. (2002) 277, 50820

1

Importin 1


Exportin 1

Poon et al. (2005) Cancer Res. 65, 7059-7064

* Goodrich et al. (1991) Cell 67:293-302

Untransformed Transformed

A) Isogenic cell line pair African Green Monkey Kidney

CV-1 COS-7 (CV-1 transformed with SV40)

B) Human osteosarcoma isogenic cell pair*

SR40 SAOS-2

(SAOS-2 with WT Rb) (Rb mutant)

C) Human skin fibroblast isogenic cell pair**

1br3 1br3N

(1br3 transformed using SV40 T-ag)

ISOGENIC CELL LINES

** From A. Lehmann (Uni. Sussex) (1999)

Curr. Biol. 9, 699-704

GFP-VP3(1-121)GFP

SAOS-2 (Transformed)

Cytoplasm

Nucleus

GFP1 33 46 82 88 97 105 111 121

LRS NLS1 NES NLS2 1-121

VP3 nuclear localization is dependent on the C-terminus

Poon et al. (2005) Cancer Res. 65,

7059-7064

GFP-VP3(1-121)GFP GFP-VP3(74-121) GFP-VP3(1-73)

GFP-VP3(1-89) GFP-VP3(1-111)

Cytoplasm

Nucleus

1-89

1-111

1-73

74-121

GFP

GFP

GFP

GFP

LRS NLS1 NES

LRS NLS1

LRS

NLS1 NES NLS2


VP3 nuclear localization is dependent on the C-terminus


7059-7064


GFP-VP3(1-89) GFP-VP3(1-111) GFP-VP3(NLS1m) GFP-VP3(NLS2m)

Cytoplasm

Nucleus

GFP LRS NES NLS2

GFP LRS NLS1 NES

NLS1m

NLS2m


VP3 nuclear localization is dependent on NLS1 and NLS2


7059-7064

GFP1 33 46 82 88 97 105 111 121

LRS NLS1 NES NLS2


GFP-VP3(1-89) GFP-VP3(1-111) GFP-VP3(NLS1m) GFP-VP3(NLS2m)

SR40 (Untransformed)

VP3 can accumulate in non-tumor cells dependent on NLS1 & NLS2

GFP1 33 46 82 88 97 105 111 121

LRS NLS1 NES NLS2


7059-7064

IMAGE ANALYSIS USING A “WORK WINDOW”:Nuclear localization of VP3(LRSm) in SR40 cells

SOFTWARE (Public Domain): “IMAGE J” (NIH)http://rsb.info.nih.gov/ij/

Trapani et al. (1996) J. Biol. Chem. 271, 4127-4133

Fn

Fn

Fc

Fn

Fb

SR40 cells – VP3(LRSm)

Fn/c = (Fn – Fb)

(Fc – Fb)

0

10

20

30

40

50

60

GFP

GFP-VP3(

1-12

1)

GFP-VP3(

74-1

21)

GFP-VP3(

1-73

)

GFP-VP3(

1-89

)

GFP-VP3(

1-11

1)

GFP-VP3(

NLS1m

)

GFP-VP3(

NLS2m

)



p < 0.0001

p < 0.0001

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

GFP1 33 46 82 88 97 105 111 121

LRS NES NLS2 1-121

Dependent on NLS1 & NLS2, VP3 accumulates to a greater extent in tumor

than in non-tumor cells


7059-7064 GFP97 105 111 121

NES NLS2 74-121NLS174 88

NLS1

82

Subcellular Localization of VP3 is Independent of Duration of Expression

DAY 1 DAY 6Fn/c

SAOS-2

SR40p = 0.0083

p = 0.0176

0

10

20

30

40

50

60

70

80

GFP

GFP-VP3(

1-12

1)

0

10

20

30

40

50

60

70

80

GFP

GFP-VP3(

1-12

1)

Is the increased nuclear accumulation in tumor cells due to more efficient nuclear

import or reduced nuclear export ?

- LMB + LMB

GFP-Rev(2-116)


GFP

GFP-VP3(1-121)

Lack of VP3 Nuclear Export Activity in Transformed Cells

LMB, leptomycin B: exportin 1 (CRM1)

inhibitor

0

10

20

30

40

50

60

70

80p < 0.0001

- LMB

+LMB

SAOS-2

GFP LRS NLS1 NES NLS2 1-121

(Transformed)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

Lack of VP3 Nuclear Export Activity in Transformed Cells

Differential Nuclear Export Activity in Transformed and Untransformed Cells

- LMB + LMB

GFP-Rev(2-116)

- LMB + LMB


GFP

GFP-VP3(1-121)


0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

p < 0.0001

p < 0.0001

- LMB

+ LMB

- LMB

+ LMB

SR40

Differential Nuclear Export Activity in Transformed and Untransformed Cells


(Untransformed)SAOS-2

(Transformed)

p < 0.0001

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

VP3 nuclear export is mediated via the C-terminal NES not the LRS

- LMB - LMB


GFP NLS1 NES NLS2 LRSm

GFP LRS NLS1 NLS2 NESm

p < 0.0001

SR40(Untransformed)

SAOS-2(Transformed)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)0

10

20

30

40

50

60

GFP-VP3(

1-12

1)

GFP-VP3(

LRSm)

GFP-VP3(

NESm)

p < 0.0001

p < 0.0001

0

10

20

30

40

50

60

GFP-VP3(

1-12

1)

GFP-VP3(

LRSm)

GFP-VP3(

NESm)

Exportin 1

Poon et al. (2005)

Cancer Res. 65, 7059-

7064

Localization of VP3 to PML NBs

Anti-PML


SR40 (Untransformed) SR40

SAOS-2

GFP-VP3(1-121) Anti-PML Merge


Poon et al. (2005) Cancer Res. 65, 7059-7064; Janssen et al. (2006) Oncogene 26, 1557-1566

Deletion/mutation of the LRS abolishes PML NB localisation

Saos2

SR40

GFP-VP3(LRSm)GFP-VP3(1-121) GFP-VP3(74-121)

LRS NLS1 NES2 NLS21 33 46 82 88 97 105 111 121

N C

LRS NLS1 NES2 NLS21 33 46 82 88 97 105 111 121

N CX

NLS1 NES2 NLS274 82 88 97 105 111 121

C

VP3 FL wt

VP3 74-121

VP3 FL LRSm

VP3 nuclear export is mediated via the C-terminal NES not the LRS

- LMB - LMB


GFP NLS1 NES NLS2 LRSm

GFP LRS NLS1 NLS2 NESm

p < 0.0001

SR40(Untransformed)

SAOS-2(Transformed)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)0

10

20

30

40

50

60

GFP-VP3(

1-12

1)

GFP-VP3(

LRSm)

GFP-VP3(

NESm)

p < 0.0001

p < 0.0001

0

10

20

30

40

50

60

GFP-VP3(

1-12

1)

GFP-VP3(

LRSm)

GFP-VP3(

NESm)Promyelocytic

leukemia (PML) nuclear bodies

Exportin 1

Poon et al. (2005)

Cancer Res. 65, 7059-

7064

Cytoplasm NucleusNuclear envelope

NPC

NPC

β1

Importin β1VP3

LRS NESNLS1 NLS2

β1GTP

Ran

GDP

RanRanGAP1

GTP

Ran

CRM1

CR

M1

GTP

Ran

Normal cell

Tumor cell

Differential Nuclear Export Activity Mediated by the VP3 NES in Normal and Tumor Cells

Testing the role of Thr108 in modulating VP3 nuclear transport

GFP LRS NLS1 NES NLS2 Glu108

GFP LRS NLS1 NES NLS2 Ala108

Ala108

Glu108


Thr108

Thr108: phosphorylated specifically in tumor cells

Rohn et al. (2002) 277, 50820

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

- LMB

+LMB

- LMB

+LMB

GFP-VP3(Ala108) - non-phosphorylatable VP3

GFP-VP3(Glu108) - mimic phosphorylated VP3

Phosphorylation at Thr108 Inhibits VP3 Nuclear Export in Tumor Cells

SR40(Untransformed)

SAOS-2(Transformed)

p < 0.0001

p < 0.0001

p < 0.0001

p < 0.0001

p = 0.032

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)

Nuc

lear

to c

ytop

lasm

ic fl

uore

scen

ce (

Fn/

c)


7059-7064

Tumor cell-specific nuclear targeting of VP3 is effected by a phosphorylation-

regulated NES Cytoplasm

Nucleus

Nuc

lea

r en

velo

pe

NPC

GDP

Ran RanGAP1

GTP

Ran

NPC

1

Importin 1

VP3 1

GTP

Ran

PML

PML

PML Nuclear Body

Exportin 1

Exp

ort

in 1

GTP

Ran

LRS NESNLS1 NLS2

Tumor cell

Normal cell

P

Poon et al. (2005) Cancer Res. 65, 7059-7064


Viral/non-viral mechanisms of drug delivery

Nucleus Cytoplasm


DNA damage

Modular Recombinant Transporter

Tumour Cell-specific Drug Targeting ?

MSHT-ag NLSHMPDTox MRT

HMP, drug (PS) binding domainDtox, Diphtheria toxin T-domain (endosomolytic)

MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake)

Rosenkranz et al. (2003) FASEB J. 17, 1121-1124

MSHVP3 tNLSHMPDToxNew MRT

Sites of photoactivation (DHFA) MRT

+ DTox

- DTox

Concentration (nM)

MRT-p6

p6

% V

iab

le c

ells

The VP3 tNTS (aa 74-121) is sufficient for tumour cell-enhanced nuclear

accumulation SR40 SAOS-2 - LMB

+ LMB

:dependent on NES activity as regulated by

negative charge at Thr108

Nucleus Cytoplasm


DNA damage

Modular Recombinant Transporter

Tumour Cell-specific Drug Targeting ?





MSHVP3 tNLSHMPDToxNew MRT


+ DTox

- DTox

Concentration (nM)

MRT-p6

p6

% V

iab

le c

ells

Anti-Cancer Approaches– Growing problem world-wide, with a need for efficient and specific anti-

cancer therapies

– Cancer cells derive from normal cells; the idea of a magic bullet to

combat cancer (a single property of a cancer cell that can be targeted

specifically) is naïve (Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50)

– VP3/apoptin – tumor cell-hyperactivity (NOT all or nothing)

– Need to combine multiple tumor cell-hyperactive components/strategies

to be able to deliver drugs to cancer cells specifically (MULTIPLICATIVE)


Non-viral approaches to achieve tumor cell-specific drug delivery

?

VP3

tNTS

MSH

Her2

c-Met

Cancer cell-specific

signallingPhosphorylatio

n

Anti-Cancer Approaches– Growing problem world-wide, with a need for efficient and specific anti-cancer therapies

– Cancer cells derive from normal cells; the idea of a magic bullet to combat cancer (a single property of a cancer cell that

can be targeted specifically) is naïve (Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50)

– VP3/apoptin – tumor cell-hyperactivity (NOT all or nothing)

– Need to combine multiple tumor cell-hyperactive components/strategies to be able to deliver drugs to

cancer cells specifically (MULTIPLICATIVE)

– VP3/apoptin – tumor cell-specific anti-cancer activities

Nuclear Signalling Lab., Dept. Biochem. & Mol.

Biol., Monash University, Clayton

IVAN POON

Cristina Oro

Gualtiero Alvisi Henna Kuusisto

Manisha Dias Kylie Wagstaff

Jingpu Zhang (Beijing)

Dept. of Mol. Genetics of Intracellular Transport,

Institute of Gene Biology, Russian Academy of

Sciences AND Dept. Biophysics, Moscow State

University

Alex Sobolev

Andrey Rosenkranz

Enhancement of cytotoxicity through cell-specific intranuclear delivery of the

photosensitiser bacteriochlorin p6 in B16-F1 melanoma cells

Concentration (nM)

p6

MRT-p6

% V

iab

le c

ells


+ DTox

- DTox





VP3/Apoptin

VP3

Nucleus

Cytoplasm





• No apoptosis

Non-isogenic cell comparisons (eg. human osteosarcoma line versus foreskin

fibroblasts)

The distinct subcellular localization of VP3 is determined by differential nuclear import/export of VP3 in tumor and normal

cells

VP3 Nuclear Accumulation is Independent of Cellular Expression Levels

GFP-VP3(74-121)

Fn/c

GFP-VP3(1-121)

0

5

10

15

20

25

30

35

40

45

50

< 20 20-40 > 40

Expression Level

Low Medium High

SAOS-2

SR40

0

5

10

15

20

25

30

35

40

45

50

< 20 20-40 > 40

Expression Level

Low Medium High

SAOS-2

SR40

Poon et al. (2005) J. Virol. 79, 1339-1341

1br3/n(transf.)

1br3

- LMB + LMB

0

55

110

165

220

Fn/c

1br3 1br3/n(transf.)

- LMB

+ LMB

Differential Nuclear Export Activity in Isogenic Transformed

and Untransformed Cells (Fibroblasts)


VP3-Induced Apoptosis

NPCNPC

NP

CN

PC

NucleusCytoplasm

Mitochondria

VP3*

NE

Transcriptionalmachinery

Apoptosis facilitating products

Hippi

Hippi Hip-1

Cytochrome crelease

Downstream caspases

Apoptosis

DNA

HipK2PML NB

PML

IMP α/β1

P

APC1APC/C

VP3*

P

ααβ1

IMPβ1

IRIGIAGITITLSL

LRS NLS1 NES NLS2

1 33 46 82 88 97 105 111 121

KPPSKKR SCDPSEYR VSKLKESLI TTT108PS RPRTAKRRIKL

N-terminus C-terminus

1 59Hippi binding

1

121

DNA binding

PML NB localization 731

80

66 DNA binding

80 1211 69Apoptosis induction Apoptosis induction

69Multimerization29

VP3

82 121APC1 binding

Domain Organization of VP3


Properties of VP3/Apoptin

VIRAL PROTEIN IMPORT REGULATION OF IMPORTANCE/ROLE IN INFECTION /EXPORT IMPORT/EXPORT

SV40 T-ag IMP dsDNA-PK/CK2 enhances import CK2 site mutants have 50% reduced viability/slowed

replication kinetics/plaque formation

Cdk site reduces import Essential for polymerase activity

Dengue NS5 IMP Hyperphospho-form in the nucleus ?? Modulate transcription/apoptosis ?? IMP1 Dengue NS3 competes IMP1 binding CRM1 CK2 reduces accumulation Blocking export early in infection increases virus production

(accelerated infection kinetics)

RSV M IMP1 Accumulation facilitated by Inhibition of transcription DNA binding ?

CRM1 CK2 enhances nuclear export Blocking export early in infection reduces virus production

CAV VP3 IMP1 Tumour cell-specific accumulation ??? Enhanced by nuclear retention

CRM1 Negative charge (phos.) at Thr108 ??? prevents export

HIV Tat Nups Cytoplasmic retention requires Nuclear localisation essential for ATP hydrolysis virus replication

? IMP1 ? Accumulation involves nuclear retention

HIV Vpr Nups Accumulation involves nuclear Role in PIC nuclear import

retention ? IMP3 ? HIV MA IMP1 Cytoplasmic retention mechanism ? Role in PIC nuclear import

REGULATION OF VIRAL PROTEIN NUCLEAR IMPORT/EXPORT

– Causes immunosuppression and severe

anemia in young chicks by inducing

apoptosis in thymocytes/erythroid

progenitors

– 3 viral proteins – VP1, VP2 and VP3

Cancer– Growing problem world-wide, with a need for

efficient and specific anti-cancer therapies

– VP3/apoptin – tumor cell-specific anti-cancer

activities

Chicken Anemia Virus (circovirus)

Properties of VP3/Apoptin

VP3

Nucleus

Cytoplasm





• No apoptosis

Transformed human embryonic kidney Rat primary hepatocytesHuman osteosarcoma cells (SAOS-2) VH10 Human foreskin fibroblasts

Documents

Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans