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in vivo animal model studies in biological science. Cancer 2. Neuroscience. Cancer research. 2. Neuroscience. Lung Cancer. Cure rate for all patients: 15%. Male. Female. EGFR Expression in NSCLC. Tumours showing high EGFR expression. - PowerPoint PPT Presentation
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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
