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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.
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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 showinghigh EGFR expression High expression associated with Poor outcome NSCLC 60-80%
EGFR signaling pathways HER HER g PLC PI3K PKC PDK-1 Ras Sos AKT PTEN Grb2 Raf p70 S6K Shc Bad/Bcl2 JAK Mek GSK3 STAT Erk Myc p27 KIP1 E2F Jun/Fos Sp1 FKHR-L1 PEA3 Elk1 CyclinD1 Citri A, et al. Exp Cell Res 2003
Molecular Targeting of EGF Receptor Cetuximab Gefitinib (IRESSA) Erlotinib (Tarceva)
Response to EGFR-TKI in NSCLC patients Initial diagnosis 2 months after gefitinib 9 months later Yu CJ. (2005) PloS Med
Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21 EGF Ligand Binding TM Tyrosine Kinase L858R (~40%) S768I (2%) R776C (2%) G719A/C (5%) L861Q (4%) 719 768 776 858 861 GxGxxG 18 K 19 20 R L 21 R 22 23 24 E746-A750 766-768 Deletions (~45%) Insertions (3%)
Modeling EGFR-TKI responses in pre-clinical model system PC9, HCC827 TKI Response(+) Response(-) ( Acquired resistance ) EGFR Mutation(+) TKI Response(-) Primary mutation(s) NSCLC TKI Response(+) EGFR Mutation(-) TKI Response(-)
Clinical response to EGFR-TKI in NSCLC patients Initial diagnosis 2 months after gefitinib 9 months later Yu CJ. (2005) PloS Med
Mechanisms of the Acquired Resistance to EGFR tyrosine kinase inhibitors in NSCLC (2008) Unknown Mechanisms T790M secondary mutation MET amplification
Modeling EGFR-TKI responses in pre-clinical model system PC9, HCC827 TKI Response(+) Response(-) ( Acquired resistance ) EGFR Mutation(+) TKI Response(-) Primary mutation(s) NSCLC TKI Response(+) EGFR Mutation(-) TKI Response(-)
Human cancer cell immunocompromised mouse xenograft model
Erlotinib treatment in PC9 orthotopic lung cancer model vehicle treated erlotinib (50mg/kg/day) treated Before administration 1 week 2 weeks 3 weeks
Data summary of erlotinib treatment in pre-clinical animal tumor xenograft model system
Multi-cycle resistance test Inoculate previously obtained in vivo drug resistant cells to second animals and subject the animals to repeated drug cycle PC9TR PC9
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 scram si-EGFR si-EGFR si-EGFR si-EGFR scram scram scram EGFR Actinin PC9 #1 #6 #10 EGFR dependent EGFR independent
Sequencing in PC9 & BR1,6,10 BR#1 PC9 I T(790) L Q I T(790) L Q BR#6 BR#10 I T(790) L Q I T(790) L Q
Protein expression and phosphorylation profile in PC9-BR clones #6 #1 #10 PC9 0 0.2 2 20 200 BIBW2992 for24H 0 0.2 2 20 200 0 0.2 2 20 200 0 0.2 2 20 200 p-EGFR EGFR p-Her3 p-MET MET p-STAT3 STAT3 p70 S6K p-AKT AKT p-ERK ERK BIM Actinin
#1 #6 #10 PC BIBW2992 treatment in inoculated mouse in vivo
invivo response to BIBW2992 (25mg/kg)
Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21 EGF Ligand Binding TM Tyrosine Kinase L858R (~40%) S768I (2%) R776C (2%) G719A/C (5%) L861Q (4%) 719 768 776 858 861 GxGxxG 18 K 19 20 R L 21 R 22 23 24 E746-A750 766-768 Deletions (~45%) Insertions (3%)
EGFR Mutations Gefitinib Responders 8/9 Non-responders 0/7 p= 0.00075 Lynch et al, NEJM 2004
Mouse EGFR non-small cell lung cancer transgenic mouse model CCSP rtTA 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
Lung cancer originated from mutant EGFR respond to various EGFR inhibitors Wong et al. (2006) Cancer Cell
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 Tumor suppressor genes Oncogenes metastasis Immune Stroma Angiogenesis WT cells
Utility of genetically-engineered mouse models of cancer early detection Genetically engineered mouse prevention Progression analysis Tumor development chemotherapy
Genetic engineering of mouse genome : knock-out and knock-in via homologous recombination
Homologous recombinant ES cell selection and blastocyst injection
Confirmation of germ line transmission and generation of knock-out(in) mouse
Conditional activation of p53 Advantages: p53 Native promoters Temporal, spatial Regulation of gene expression unbiased
P53 LSL/LSL is a phenocopy of p53-/- Cre-recombinase-Oestrogen-Receptor-T2
p53 reactivation mouse model study with conditional gene expression mouse • p53 inactivation is required for the maintenance of p53 mutant tumors • p53 gene delivery or other ways to reactivate p53 in p53 mutant tumor • could be a therapeutic option