Applications of Mouse Models

To Cancer Research

Jeff Green, M.D.

Chief, Transgenic Oncogenesis and
Genomics Section
Laboratory of Cancer Biology and
Genetics
NCI, Bethesda, MD
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Growth Hormone Transgenic Mice

Genetically Engineered
Mice:
Limited only by our
imagination!
 Topics:
• advantages/disadvantages
of mouse
models
• designing and generating
models
• validating models as
surrogates for
human studies
• applying models for pre-
clinical
 Why Mice?
• They don’t sue!
• No IRB (but ACUC!)
• Human trials are expensive
• Human trials take a long time
• Genetic background
• Can identify modifier genes
• Can control conditions precisely
• Can validate a target
• Can generate complex genetic models
• Can study stage-specific effects of
intervention
• Can easily perform combination studies
Advantages of GEM Over

In vitro Approaches
• Study gene in context of entire animal
• In the context of an intact immune
system
• Effects on animal development
• Study cancer stage progression
• Study tissue-tissue interactions
• Study genetic interactions
• Identify modifier loci
• Tools for pre-clinical testing
 Disadvantages:
• A mouse is only a mouse!
• Targeting to correct
compartment
• Hormone-responsiveness of
promoters
• Developmental timing
• Expression levels of transgene
• Pharmacology
• Tissue composition
• Poor models of metastases
 Environment Genetic Background
• chemical exposures
• race
• radiation/UV exposure
• genetic modifiers
• viral infections
• familial cancer syndromes
• immune suppression
Cancer
Aging Risk Prior cancer history

Somatic Gene
Life style
Dysregulation/Muta
• diet/alcohol
•Oncogene tion
• exercise
activation/over-expression
• obesity
• Loss of suppressor gene
• mentstrual history/parity history
function
• Chromosomal/gene
rearrangement
Intelligent Design

Creating mice in our own

molecular image
Oncogenic Targets of GEM Mammary CA Models

Growth Factors/Receptors
Steroid Hormone
Signaling IGF
TGF β
Her2/neu
Aromatase PyMT IGFR TGFβR
Estrogen
Androgen ER PIP-2

Retinoids AR
PIP-3
PI3K JAK
RAS
SMADs
Vitamin D RXR PKC MEK STAT
ERK
VDR AKT
PTEN

Inflammatory
Prostoglandins COX-2 Response

BRCA1
Transcriptional
Regulation CELL
myc CYCLE

p53 TAG
Rb
 Oncogenesis
In GEM

?
Genetic Aberration(s)
(~All cells) Cancer

- p53
- BRCA1 ONCOGENES:
Her2/neu
Myc
Ras
PyMT
SV40 Tag P53; Rb
? 
 Mouse Cancers Human Cancers

Biology

Histopathology

Therapeutic Trials Genomic Analyses Therapeutic Trials

Expression Profiling

Bioinformatics

Model Validation
Transgenic Targeting to the
Mammary and Prostate Glands

• tissue-specificity
• cell-compartment specificity
• developmental timing
• hormone-independent promoter
Hormone

Promoter Oncogene
BAC Recombineering
STRATEGIES FOR HOMOLOGOUS RECOMBINATION

NEO TK

II III

II III

Insertion/Selection

NEO

II III
 NEO TK

II III

II III

Deletion/Selection

NEO
KNOCK-IN STRATEGY

NEO * TK

II III

II III

Replacement

NEO *
II III
CONDITONAL KNOCK-0UT (Cre-lox)

NEO L L TK

II III

II III

Replacement/Selection

NEO L L

II III
 NEO L L

II III

X
cre recombinase
Promoter

II

NEO L

III
Examples of Useful
Cancer Models

Brain
Ovary
Prostate
Breast
Lung
Colon
Pancreas
Ras + Akt Glioma Model

(E. Holland)
Ovarian Cancer Model
(S. Orsulic)
 C3(1)/Tag Transgenic Model
Mammary Lesion
Prostate Tumor Progression Progression

N PIN CA

Human Mouse

ATG
C3(1)/Tag
I SV40 Tag
*
Tumor Progression in Breast Cance

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10% Apoptosis

0% Shibata, EMBO, 2001

↑ Bax

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Mouse

Normal DCIS/MIN Invasive CA

Pancreatic Cancer
(D. Tuveson)
Prostate

Courtesy of N. Greenberg
 Lung
(T. Jacks)
Colon
 Additional GEM
Technologies

• Viral delivery systems

(retrovirus/lentevirus)
• RACS system
• Embryonic reconstitution
• Chimeric mice (VEGF; ets
K.O.s)
 Embryonic Reconstitution

Embryonic U.G.
Sinus
Retrovirus + ras/myc
 Y.Li
The RCAS-TVA Technology

RCAS - a viral vector derived from ALV-A

TVA - avian receptor for ALV-A

SA

SA
SD

LTR gag pol env LTR

X
gag, pol
env
gene X
TVA
ene Delivery with RCAS Vector
tv-a
Pr TV-a transgeni
c mouse

LTR gag pol env LTR

 active oncogenes
 DN TSGs
 inducible genes
 Cre recombinase
 Tet activator
 marker genes
Conditional Expression
Models:

• Control of tissue specificity

and
developmental timing of gene
expression.

• Study oncogene dependence.

 Tissue­Specific 
Promoter TK or CD

TISSUE SPECIFIC ABLATION 
TISSUE SPECIFIC ABLATION
WITH DRUG
WITH DRUG

Tissue­Specific 
Promoter Tet­transactivator protein (on/off)

Tet­operator  
Promoter Gene of Interest

TEMPORAL CONTROL OF GENE EXPRESSION 
TEMPORAL CONTROL OF GENE EXPRESSION
WITH TETRACYCLINE
WITH TETRACYCLINE
Oncogene Dependence of
Tumors
In vivo Imaging
GFP

LUC
Advanced In Vivo Imaging Techniques
Choosing the right
model
What is your question?
evelopment of Pathology Nomenclature fo
Mouse Models of Mammary Cancer

• need to standardize nomenclature

• need to translate between human and mouse
pathology
• need to assess similarities/differences
between human and
mouse
(Cardiff et al., Oncogene, 2000.)
 Comparing Genomic
Aberations Between Mouse
Models and
Human Cancers
(Chin L and Depinho R)
 How Can the Mouse Help Us Better
Understand Breast Cancer?

elp sift through molecular chao

Integrating the Mouse
with
Human Genomic Studies

• identify oncogenic
signatures.
• improve identification of
biologic
networks through
identification
of functionally conserved
genes.
 

Enormous Defined
Genetic Diversity Genetic Background
 

Global Expression Profiiling of Tumors


 

Evolutionarily Conserved
Genetic Networks
High Throughput Microarray
Sorlie et al., PNAS (2001
 ER- stem cell Progenitor cell

ER- ER- ER+

C3(1)/Tag MMTV-her2/neu P53-/-

P53-/- MMTV-ras
BRCA1-/-;p53+/- MMTV-myc
MMTV-PyMT
 Common Genes/Biomarkers
Related to Similar Biologic State

ER+

Mouse

ER+

Human
The Mouse-Human Classifier is the Best Predictor of
ER Status

Human classifier
Mouse/Human classifie
Mouse classifier

Training Test
Mouse/Human
Classifier
ER+
ER-
Using the model for
pre-clinical testing
Inhibition of Mammary Tumor Progression
By Recombinant Endostatin

Control

Endostatin
 Lizt, 2001

Flk-1/DKR

Angiogenes
is
 VEGF Receptor
Inhibitors GSK: J. Stafford and
Rakesh Kumar

• GW2286 as prototype receptor kinase inhibitor

• Specific inhibitor of KDR phosphorylation/Flk-1 (VEGFR-2).

H3C

Enzyme hVEGFR2 mVEGFR2 src EGFR CDK2

N
N OMe
IC50 (nM) 8 48 758 7,079 1,622
H NH
OMe
N

N N OMe
H

• Inhibits VEGF-induced endothelial cell proliferation and

KDR phosphorylation.

• Inhibits tumor vascular density, and Matrigel plug and

• cornea micropocket assays.

no toxicity
 Inhibition of tumor
growth
with VEGFRII inhibitor
Survival (percentage)
100 Control
10 mg
100 mg
75
Survival

50

25

0
16 18 20 22 24 26
Age (wk)

Huh, J. et al. 2005, Oncogene

INITIATION PROGRESSION

Dormant

Mammary  Invasive CA
Intraepithelial  Metastasis
Neoplasia

At what stages do preventive agents

inhibit cancer?
C3(1)/Tag Mammary Histopatholog
P53
Rb A B C
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Mouse
8-12 weeks 17+ weeks
Normal DCIS/MIN Invasive CA

“Chemoprevention”
DHEA
DFMO
9-cRA
Endostatin
The Metastatic Process

dormancy metastasis
1. Genetic
2. Epigenetic-intracellular
-extracellular:ECM

Chambers AF (2002):Nat Rev Cancer 8:563-

 MCF-7 cells are dormant in vivo

MDA-MB-231
2 weeks
post injection

MCF-7
9 weeks
post injection

SCOM H&E
 Experimental Alterations
of Risk Factors

• Energy balance
Generation of GEM • Nutritional composition
Models With Genetic • Hormone exposure
Changes Relevant to • Carcinogens
Human Cancer • Pregnancy (breast)
• Infectious agents
• UV exposure (skin) Determining
•Radiation Predictive
•Genetic Background Value of GEM
Models

Evaluation of Model Selection of Assessment of Response Clinical Trials

Phenotype: Candidate Models in Models In Humans

• Disease natural history • Tumor latency

• Histopathology • Tumor multiplicity
• Hormone responsiveness • Growth rate
(breast and prostate) ADMINISTRATION OF • Metastases
• Cell of origin PREVENTIVE AGENT(S) • Survival
• Genome integrity • Histologic changes
• Gene expression • Gene expression
• Protein markers • Biomarkers

Functional conservation
of genes/networks

Green and Hudson, Nature Reviews Cancer, 2005

Lab Members (current/former)

Dalit Barkan
Christina Bennett
Alfonso Calvo
Isabel Chu
Kartiki Desai
Tamaro Hudson
Jung-im Huh
Mark Hoenerhoff
Claudine Kavanaugh
Kristin Kee
Hark Kim
Zi-yao Liu
Aleksandra Michalowska
Ting Qiu
Masa-aki Shibata
Christy Tomlinson
Min Zhu