CONDITIONAL KNOCKOUT OF P53 IN MESENCHYMAL CELLS OF MICE RESULTS IN OSTEOSARCOMAS

1. CONDITIONAL KNOCKOUT OF P53 IN MESENCHYMAL CELLS OF MICE RESULTS IN OSTEOSARCOMAS PATRICK P. LIN, FENGHUA JIN, GUILLERMINO LOZANO November 13, 2004 Montreal, Canada

2. Introduction

3. 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

4. Li-Fraumeni SyndromeGerm-Line P53 Mutation Hisada, JNCI 90:606, 1998

5. 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

6. P53 Deficient Mice Homozygous knockout Heterozygous knockout Jacks et al, Current Biology 4:1, 1994 Donehower, Current Biology 7:296, 1996

7. Rb • Another important tumor suppressor gene • Involved in cell cycle control

8. Hereditary RetinoblastomaGermline Mutation of Rb • Osteosarcoma 2nd most common malignancy • Rb - mutations found in sporadic osteosarcomas also

9. Rb Knockout Mice • Homozygous mice are embryonic lethal • Heterozygous mice only develop pituitary tumors • No osteosarcomas or other sarcomas

10. Goal of This Study • Develop genetic mouse model of sarcomas • Effect of specific mutations on tumor phenotype • Tumor suppressor genes

11. 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

12. 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 cre loxP gene

13. 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

14. Prx1 • Paired-related homeobox gene • Previously called Mhox • Regulates embryonic development • Prx1 Knockout mice • Craniofacial defects • Limb shortening • Spina bifida

15. Prx1 Expression in Embryoes • Primarily in Limbs & Cranial Mesenchyme Martin, Genesis 26:225, 2000

16. 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

17. 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

18. Prx1-Cre Expression • Prx1-Cre is expressed primarily in limbs • Cross to R26R (Rosa26 lacZ reporter)

19. 2 3 4 5 6 7 8 9 10 11 1 lox lox P53lox and Rblox Mice P53lox Exon 19 Rblox lox lox Marino, Genes & Dev 14:994, 2000

20. Results

21. Tumor Cohorts

22. Prx1Cre – mediated knockout of p53 p=0.008

23. Cause of DeathPrx1cre P53 lox/lox

24. Osteosarcomamouse 1296age 39 wksfemur

25. Location of OsteosarcomaProximal Femur Predominant Site

26. Mouse 1304age 40 wksscapula

27. Genotype of Tumor Neg control Tumor Tail • PCR verifies loss of floxed P53 gene in tumor

28. Osteoblastic Osteosarcoma

29. Osteosarcoma Metastasis Prx1creP53 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

30. 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

31. Soft Tissue Sarcoma • Poorly differentiated, unclassified soft tissue sarcoma • No osteoid • No involvement of bone

32. Lymphoma of Bone • Rarely seen • Arose in bone • Thymus not involved

33. 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!

34. 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

35. Discussion

36. Preliminary Study • Establishes framework for future studies • Larger numbers of mice needed to corroborate initial findings here

37. 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

38. 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

39. 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

40. 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

41. Acknowledgements • Orthopaedic Research & Education Foundation Grant (02-026) • Anton Berns • John Parant • Jim Martin • Carolyn Van Pelt • Richard Behringer • Victor Olnichikov

42. Thank You