CONDITIONAL KNOCKOUT OF P53 IN MESENCHYMAL CELLS OF MICE

RESULTS IN OSTEOSARCOMAS

PATRICK P. LIN, FENGHUA JIN, GUILLERMINO LOZANO

November 13, 2004Montreal, 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 SyndromeGerm-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

Heterozygousknockout

Homozygousknockout

Jacks et al, Current Biology 4:1, 1994Donehower, Current Biology 7:296, 1996

Rb

• Another important tumor suppressor gene

• Involved in cell cycle control

Hereditary RetinoblastomaGermline 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

creloxP loxP loxP

gene

Published WorkRb/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)

P53lox and Rblox Mice

loxlox

1 2 3 4 5 6 7 8 9 10 11

P53lox

Rblox

Exon 19

loxlox

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

Time (wks)

120100806040200

Ove

rall

Su

rviv

al

1.0

.8

.6

.4

.2

0.0

p53-lox/lox

p53-lox/w t

p=0.008

Cause of DeathPrx1cre P53 lox/lox

Osteosarcoma

Soft tissue sarcoma

Non-tumor

Lymphoma

Osteosarcomamouse 1296 age 39 wks

femur

Location of Osteosarcoma Proximal Femur Predominant Site

Mouse 1304age 40 wks

scapula

Genotype of Tumor

• PCR verifies loss of floxed P53 gene in tumor

Tail

Tumo

rNeg

contr

ol

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 KnockoutRblox/lox and Rblox/null

• No developmental abnormalities– Limbs normal

– Mice fertile

• No sarcomas– Most die of old age

– 1 tumor (thyroid)

• Rb knockout does not initiate sarcoma formation in mice! Time (wks)

100806040200

Ove

rall

Su

rviv

al

1.0

.8

.6

.4

.2

0.0

Rb lox/null

Rb lox/lox

Rb Knockout Accelerates Sarcoma Formation

• Simultaneous conditional knockout of p53 and Rb

• p < 0.0001

• Preliminary data

– Utilizes col2A1cre mouse to achieve double conditional knockout

Weeks

80706050403020100

Su

rviv

al

1.0

.8

.6

.4

.2

0.0

col2A1crep53lox/lox Rblox/lox

prx1crep53lox/lox

Weeks

80706050403020100

Su

rviv

al

1.0

.8

.6

.4

.2

0.0

col2A1crep53lox/lox Rblox/lox

prx1crep53lox/lox

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