in vivo animal model studies in biological science

in vivo animal model studies in biological science

1. Cancer

2. Neuroscience

1. Cancer research

2. Neuroscience

Lung Cancer

• Cure rate for all patients: 15%

Male Female

EGFR Expression in NSCLC

NSCLC 60-80%

Tumours showinghigh EGFR expression

Poor outcome

High expression associated with

EGFR signaling pathways

HER HER

PLC g PI3K

Shc

SosRas

Raf

Mek

PKC

AKT

CyclinD1Elk1

MycSp1

PDK-1

p70S6K

PTEN

FKHR-L1

JAK

STAT

Grb2

p27KIP1Jun/Fos E2F

PEA3

Bad/Bcl2

GSK3

Erk

Citri A, et al. Exp Cell Res 2003

Molecular Targeting of EGF Receptor

Gefitinib (IRESSA) Erlotinib (Tarceva)

Cetuximab

Response to EGFR-TKI in NSCLC patients

Yu CJ. (2005) PloS Med

Initial diagnosis 2 months after gefitinib 9 months later

GxGxxG K

E746-A750

R

768

L R

858

719

G719A/C (5%)

861

L858R (~40%) L861Q (4%)

18 19 20 21 22 23 24

Deletions (~45%)

766-768

Insertions (3%)

776

S768I (2%) R776C (2%)

EGF Ligand Binding

Tyrosine Kinase

TM

Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21

NSCLCNSCLC

EGFR Mutation(+)

EGFR Mutation(-)

TKIResponse(+)

TKI Response(-)

TKI Response(+)

TKI Response(-)

Response(-)

Primary mutation(s)

( Acquired resistance )

Modeling EGFR-TKI responses in pre-clinical model system

PC9, HCC827

Clinical response to EGFR-TKI in NSCLC patients

Yu CJ. (2005) PloS Med

Initial diagnosis 2 months after gefitinib 9 months later

MET amplification

T790M secondary mutationUnknown Mechanisms

Mechanisms of the Acquired Resistance

to EGFR tyrosine kinase inhibitors in NSCLC (2008)

NSCLCNSCLC

EGFR Mutation(+)

EGFR Mutation(-)

TKIResponse(+)

TKI Response(-)

TKI Response(+)

TKI Response(-)

Response(-)

Primary mutation(s)

( Acquired resistance )

Modeling EGFR-TKI responses in pre-clinical model system

PC9, HCC827

Human cancer cell immunocompromised mouse xenograft model

Erlotinib treatment in PC9 orthotopic lung cancer model

Before administration 1 week

2 weeks 3 weeks

vehicle

erlotinib (50mg/kg/day)

treated

treated

Tumorigenic animals

Treatment duration

Culture succeeded

Tumor regression

Culture failed

Continue treatment

PC9 lung 8 6 months 3 1 4 0

PC9 S. C. 13 5 months 1 0 3 9

HCC827 lung 4 3 months 0 4 0 0

HCC827 S. C. 1 3 months 0 0 0 1

Data summary of erlotinib treatment in pre-clinical animal tumor xenograft model system

PC9TRPC9

Multi-cycle resistance test

Inoculate previously obtained in vivo drug resistant cells tosecond animals and subject the animals to repeated drug cycle

1st generation EGFR-TKI

gefitinib, erlotinb : reversible EGFR blocker

2nd generation EGFR-TKI

e.g. pan-erbB blocker, multi-target EGFR blocker, irreversible EGFR blocker

(BIBW2992)

The Effect on Cell Viability of si-EGFR in PC-BR clones

EGFR

Actinin

scra

msc

ram

scra

msc

ram

si-EGFR

si-EGFR

si-EGFR

si-EGFR

PC9 #10#6#1

EGFR dependentEGFR independent

Q L I T(790)

PC9

Sequencing in PC9 & BR1,6,10

Q L I T(790)

BR#1

Q L I T(790)

BR#6Q L I T(790)

BR#10

Protein expression and phosphorylation profile

in PC9-BR clones

EGFR

Actinin

AKT

ERK

p-EGFR

p-AKT

p-Her3

p-ERK

STAT3

METp-MET

p-STAT3

p70 S6K

BIM

0 0.2 2 20 200

0 0.2 2 20 200 BIBW2992 for24H

0 0.2 2 20 200

PC9 #10#6 0 0.2 2 20 200

#1

20

BIBW2992 treatment in inoculated mouse in vivo

#1 #6

PC #10

in vivo response to BIBW2992

(25mg/kg)

GxGxxG K

E746-A750

R

768

L R

858

719

G719A/C (5%)

861

L858R (~40%) L861Q (4%)

18 19 20 21 22 23 24

Deletions (~45%)

766-768

Insertions (3%)

776

S768I (2%) R776C (2%)

EGF Ligand Binding

Tyrosine Kinase

TM

Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21

EGFR Mutations

Gefitinib Responders 8/9

Non-responders 0/7 p= 0.00075

Lynch et al, NEJM 2004

Construction of transgenic mouse model

CCSP rtTA

Wong et al. (2006) Cancer Cell

Mouse EGFR non-small cell lung cancer transgenic mouse model

Wong et al. (2006) Cancer Cell

Tet-inducible mutant EGFR expression in mouse lung

Wong et al. (2006) Cancer Cell

EGFR mutation is oncogenic

Wong et al. (2006) Cancer Cell

EGFR expression is required for the maintenance of tumor

Wong et al. (2006) Cancer Cell

Wong et al. (2006) Cancer Cell

Lung cancer originated from mutant EGFR respond to various EGFR inhibitors

Transgenic mutant EGFR animal model study

1. mutant EGFR is oncogenic

2. continued expression of EGFR is required for the maintenance of tumor

3. mutant EGFR is a therapeutic target

Factors controlling tumorigenesis

Immune

Oncogenes Tumor suppressor genes

Stroma Angiogenesis

WT cells

metastasis

Utility of genetically-engineered mouse models of cancer

Geneticallyengineeredmouse

Tumor development

Progression analysis

early detection

prevention

chemotherapy

Genetic engineering of mouse genome : knock-out and knock-in via homologous recombination

Embryonic stem cell culture

Homologous recombinant ES cell selection and blastocyst injection

Generation of chimera mouse

Confirmation of germ line transmission and generation of knock-out(in) mouse

Conditional activation of p53

Advantages:

p53Native promoters Temporal, spatialRegulation of gene expressionunbiased

Conditional knock-out system

P53 LSL/LSL is a phenocopy of p53-/-

Cre-recombinase-Oestrogen-Receptor-T2

p53 reactivation mouse model study with conditional gene expression mouse

1. p53 inactivation is required for the maintenance of p53 mutant tumors

2. p53 gene delivery or other ways to reactivate p53 in p53 mutant tumor could be a therapeutic option