Download Session 10 - Patrick Lin

CONDITIONAL KNOCKOUT OF P53 IN
MESENCHYMAL CELLS OF MICE
RESULTS IN OSTEOSARCOMAS
PATRICK P. LIN, FENGHUA JIN,
GUILLERMINO LOZANO
November 13, 2004
Montreal, Canada
Introduction
P53
• Tumor suppressor gene
– Most commonly mutated tumor suppressor
– Approximately 50% of all cancers
• Guardian of the genome
– Activated in response to DNA damage
– Cell cycle arrest
– Apoptosis
Li-Fraumeni Syndrome
Germ-Line P53 Mutation
Hisada, JNCI 90:606, 1998
P53 Knockout Mice
• Developed in early 1990s
• Mice exhibit a wide spectrum of tumors
• Problem
– Homozygous knockout die of lymphomas rapidly
• Within 6 months
• Relatively few sarcomas
– Heterozygous knockout produces sarcomas slowly
• Usually about 18 months
P53 Deficient Mice
Homozygous
knockout
Heterozygous
knockout
Jacks et al, Current Biology 4:1, 1994
Donehower, Current Biology 7:296, 1996
Rb
• Another important tumor suppressor gene
• Involved in cell cycle control
Hereditary Retinoblastoma
Germline Mutation of Rb
• Osteosarcoma 2nd most common malignancy
• Rb - mutations found in sporadic osteosarcomas
also
Rb Knockout Mice
• Homozygous mice are embryonic lethal
• Heterozygous mice only develop pituitary tumors
– No osteosarcomas or other sarcomas
Goal of This Study
• Develop genetic mouse model of sarcomas
– Effect of specific mutations on tumor phenotype
• Tumor suppressor genes
Strategy
• Conditional knockout of tumor suppressor genes
– Early knockout
• Less differentiated cells more likely to give rise to tumors
– Only in mesenchymal tissue
• Bone, cartilage, muscle, connective tissue
Cre-Lox Recombination
• Cre recombinase
– Transgenic mouse expresses Cre under the control of a specific
promoter
• LoxP sites
– Short 34 bp sequence recognized by Cre
– Introduce into mouse genome around targeted gene
loxP
loxP
gene
cre
loxP
Published Work
Rb/P53 Conditional Knockout in Brain
• GFAP-Cre x P53lox/lox Rblox/lox
– 100% medulloblastomas
• within 4 months
• GFAP (glial fibrillary acidic protein) – expressed only in brain
Marino et al, Genes & Dev 14:994, 2000
Prx1
• Paired-related homeobox gene
– Previously called Mhox
– Regulates embryonic development
• Prx1 Knockout mice
– Craniofacial defects
– Limb shortening
– Spina bifida
Prx1 Expression in Embryoes
• Primarily in Limbs &
Cranial Mesenchyme
Martin, Genesis 26:225, 2000
Prx1 Expression in Embryonic Tissue
• Not in epithelium
– (e & ep, panels B,D)
• Only in mesoderm
– Condensing mesenchyme
of limb bud (cm, panel C)
– Mesenchyme (m, panel D)
– Periosteum (p, panels E,F)
– Maxillary process (mp,
panels A,B)
Martin, Genesis 26:225, 2000
Prx1-Cre Transgenic Mouse
• Utilizes 2.4 kb fragment
– 5’ genomic flanking region of Prx1 gene
– contains 530 bp core fragment of Prx1 promoter
Logan, Genesis 33:77, 2002
Prx1-Cre Expression
• Prx1-Cre is expressed primarily in limbs
– Cross to R26R (Rosa26 lacZ reporter)
lox
P53
and
lox
Rb Mice
234
1
5 6 7 89
10
11
P53lox
lox
lox
Exon 19
Rblox
lox
lox
Marino, Genes & Dev 14:994, 2000
Results
Tumor Cohorts
Cohort
#Mice
Age
(wks)
Tumors
Prx1-Cre+/- Rblox/lox
13
44-98
1
Prx1-Cre+/- Rblox/NULL
8
44-98
0
Prx1-Cre+/- P53lox/WT
23
33-96
4
Prx1-Cre+/- P53lox/lox
25
42-68
18
Prx1Cre – mediated knockout of p53
1.0
.8
p53-lox/w t
.6
.4
p53-lox/lox
p=0.008
.2
0.0
0
20
40
Time (wks)
60
80
100
120
Cause of Death
Prx1cre P53 lox/lox
Soft tissue
sarcoma
Lymphoma
Osteosarcoma
Non-tumor
Osteosarcoma
mouse 1296
age 39 wks
femur
Location of Osteosarcoma
Proximal Femur Predominant Site
scapula
8%
spine
17%
tibia
8%
distal femur
8%
prox femur
59%
Mouse 1304
age 40 wks
scapula
Genotype of Tumor
• PCR verifies loss of
floxed P53 gene in tumor
Osteoblastic Osteosarcoma
Osteosarcoma Metastasis
Prx1cre P53 lox/lox
• Lung: 2 of 12 (17%) mice
– Visible on Xray or necropsy
– Microscopic mets not easily
detectable
– Primary tumor grows
extremely fast (days)
• Bone: 4 of 12 tumors
• Spleen: 1 of 12 tumors
Metastatic vs. Multifocal Disease?
• 4 of 12 (33%) mice
• Osteoblastic tumors in
multiple bones
• Multifocal disease vs. mets?
– 1 mouse had both lung and
bone mets
Soft Tissue Sarcoma
• Poorly differentiated, unclassified
soft tissue sarcoma
• No osteoid
• No involvement of bone
Lymphoma of Bone
• Rarely seen
• Arose in bone
• Thymus not involved
Rb Conditional Knockout
Rblox/lox and Rblox/null
• No developmental
abnormalities
1.0
Rb lox/lox
.8
– Limbs normal
– Mice fertile
.6
• No sarcomas
.4
Overall Survival
– Most die of old age
– 1 tumor (thyroid)
Rb lox/null
• Rb knockout does not initiate
sarcoma formation in mice!
.2
0.0
0
Time (wks)
20
40
60
80
100
Rb Knockout Accelerates Sarcoma
Formation
• Simultaneous conditional
knockout of p53 and Rb
1.0
.8
prx1cre
p53lox/lox
• p < 0.0001
• Preliminary data
– Utilizes col2A1cre mouse
to achieve double
conditional knockout
.6
.4
col2A1cre
p53lox/lox Rblox/lox
.2
0.0
0
10
Weeks
20
30
40
50
60
70
80
Discussion
Preliminary Study
• Establishes framework for future studies
• Larger numbers of mice needed to corroborate
initial findings here
Mice Tend to Make Osteosarcomas
• Distribution of tumors is not random
– Histology - osteosarcomas
– Location - femur
– Not predicted
• Prx1-cre knock outs p53 in the limb bud
• All mesenchymal tissues in limb should have equal chance of tumor
formation
• There must be strong genetic & developmental influences
that favors osteosarcoma in femur
P53 Conditional Knockout
• Good strategy to create sarcomas
– Eliminate nearly all other tumors
• Occasional lymphoma of bone occurs
– Confirms hypothesis that mice bearing homozygous deletion of
p53 will almost always develop sarcomas
• P53 mutation is an initiating event for sarcomas
– Represents one important pathway for sarcoma formation
– This is generally not believed to be true for carcinomas
Rb Conditional Knockout
• No developmental defects (surprisingly)
– Note global Rb knockout is lethal in embryoes
• Poor initiator of sarcoma formation in mice
– No tumors with Rb knockout alone
• Accelerates sarcoma formation
– Double knockout of p53 & Rb produces sarcomas
more rapidly
Conclusions
• Conditional knockout of p53 produces sarcomas
– Majority are osteoblastic osteosarcomas of femur
– Poorly differentiated soft tissue sarcomas also form
• Conditional knockout of Rb accelerates sarcoma
formation
– Does not initiate sarcoma formation in mice
Acknowledgements
• Orthopaedic Research &
Education Foundation
Grant (02-026)
• Anton Berns
• John Parant
• Jim Martin
• Carolyn Van Pelt
• Richard Behringer
• Victor Olnichikov
Thank You