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Modeling human osteosarcoma in the mouse: From bedside to bench
Bone 47 (2010) 859–865
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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Review
Modeling human osteosarcoma in the mouse: From bedside to bench Katherine A. Janeway a,⁎, Carl R. Walkley b,⁎ a
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, 44 Binney St, Boston, MA 02115, USA b St. Vincent's Institute of Medical Research & Department of Medicine, University of Melbourne, 9 Princes St, Fitzroy, Victoria 3065, Australia
a r t i c l e
i n f o
Article history: Received 7 June 2010 Revised 28 July 2010 Accepted 30 July 2010 Available online 6 August 2010 Edited by: T. Jack Martin Keywords: Osteosarcoma Mouse model Bone biology Cancer
a b s t r a c t Osteosarcoma (OS) is the most common primary tumour of bone, occurring predominantly in the second decade of life. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized (non-metastatic) disease approaching 70%. At presentation approximately 20% of patients have metastases and almost all patients with recurrent OS have metastatic disease and cure rates for patients with metastatic or recurrent disease remain poor (b 20% survival). Over the past 20 years, considerable progress has been made in the understanding of OS pathogenesis, yet these insights have not translated into substantial therapeutic advances and clinical outcomes. Further progress is essential in order to develop molecularly based therapies that target both primary lesions as well as metastatic disease. The increasing sophistication with which gene expression can be modulated in the mouse, both positively and negatively in addition to temporally, has allowed for the recent generation of more faithful OS models than have previously been available. These murine OS models can recapitulate all aspects of the disease process, from initiation and establishment to invasion and dissemination to distant sites. The development and utilisation of murine models that faithfully recapitulate human osteosarcoma, complementing existing approaches using human and canine disease, holds significant promise in furthering our understanding of the genetic basis of the disease and, more critically, in advancing pre-clinical studies aimed at the rational development and trialing of new therapeutic approaches. © 2010 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges in the clinical study of human osteosarcoma . . . . . . . . . . . . . Human osteosarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic susceptibility to OS in humans. . . . . . . . . . . . . . . . . . . . Pathophysiology of human OS . . . . . . . . . . . . . . . . . . . . . . . . Animal models of osteosarcoma . . . . . . . . . . . . . . . . . . . . . . . . . Genetically engineered mouse models of human osteosarcoma . . . . . . . . . . Generation of osteo-lineage specific models with high penetrance of osteosarcoma . Refinements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of murine models . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction
⁎ Corresponding authors. Fax: +61 3 9416 2676. E-mail addresses: [email protected] (K.A. Janeway), [email protected] (C.R. Walkley). 8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2010.07.028
Osteosarcoma (OS) is the most common primary tumour of bone, occurring predominantly in the second decade of life. It is the most frequently occurring paediatric non-hematological tumour of bone and the 5th most prevalent malignancy of adolescence [1]. OS is
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notable for its locally aggressive behaviour and early metastasis formation. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized (non-metastatic) disease approaching 70%, but at the cost of considerable therapy-related morbidity [2,3]. At presentation approximately 20% of patients have metastases and almost all patients with recurrent OS have metastatic disease. Cure rates for patients with metastatic or recurrent disease remain poor (b20% survival) [4,5]. Over the past 20 years, considerable progress has been made in the understanding of OS pathogenesis. These insights have not yet been translated into substantial therapeutic advances and clinical benefit. Further progress is essential in order to develop molecularly based therapies that target both primary lesions as well as metastatic disease. The development and utilisation of animal models that faithfully recapitulate human osteosarcoma holds significant promise in furthering our understanding of the genetic basis of the disease and, more critically, in advancing pre-clinical studies aimed at the trialing and rational development of new therapeutic approaches. Challenges in the clinical study of human osteosarcoma Despite being the most common primary tumor of bone, osteosarcoma is relatively rare with approximately 600 patients developing osteosarcoma each year in the United States [6]. Osteosarcoma occurs most often in adolescents and young adults, a patient population that is less likely to enrol on clinical trials [7]. Due to these factors, national, multi-institutional phase III trials in osteosarcoma typically require at least 4 years of patient enrolment, making rational selection of drugs for study in phase III trials essential [8]. However, selecting drugs to study in phase III trials in osteosarcoma is complicated by several factors. Because of the bony matrix, even when exposed to highly effective therapy, osteosarcoma may not demonstrate a radiographic response [9]. Due to the complexity of osteosarcoma biology, research has, so far, led to only a limited number of clinically relevant biologic insights [10]. In addition, standard of care for many recurrent osteosarcomas is surgical resection, complicating the conduct of phase II clinical trials in osteosarcoma. Due to these and other factors, there has been only one significant therapeutic advance in osteosarcoma in the past 20 years, the addition of Liposomal Muramyl-Tripeptide Phosphatidyl Ethanolamine (L-MTP-PE) to standard upfront chemotherapy resulting in a modest improvement in overall survival [8]. Given the considerable challenges inherent in clinical drug development for osteosarcoma, novel approaches to drug and drug target discovery, with particular attention to drug development for metastatic osteosarcoma, are needed. Human osteosarcoma Genetic susceptibility to OS in humans Familial cancer syndromes, whilst rare, have provided important insight in to the underlying genetics of human disease. With respect to OS, three cancer predisposition syndromes stand out as significantly increasing the incidence of OS. These are Li–Fraumeni syndrome [11– 15], hereditary retinoblastoma [16–19] and Rothmund–Thomson and other syndromes associated with mutations in the RECQL DNA helicases [20–23]. Li–Fraumeni syndrome is due to germ-line heterozygous loss of function mutations in p53 [24]. Li–Fraumeni syndrome predisposes affected individuals to a wide variety of cancers, most commonly breast, brain tumors, leukemia, and bone and soft tissue sarcomas. Approximately 10% of malignancies occurring in patients with Li– Fraumeni syndrome are osteosarcoma [25]. Individuals with hereditary retinoblastoma carry a germline deletion or inactivating mutation in one allele of the retinoblastoma 1 gene (Rb) and develop
retinoblastoma, a malignant tumor of the retina, with a penetrance of 90%. Approximately 50% of the malignancies other than retinoblastoma developing in individuals with hereditary retinoblastoma are osteosarcomas [26]. Rothmund–Thomson syndrome is an autosomal recessive cancer predisposition syndrome caused by germline mutations of RECQL4, a DNA helicase [27,28]. This syndrome is characterized by sparse hair growth, skin rash, skeletal dysplasia and an increased risk of osteosarcoma with 30% of patients with Rothmund– Thomson syndrome developing osteosarcoma. Werner and Bloom syndromes are related to Rothmund–Thomson in that they are caused by mutations in RECQ DNA helicases, WRN and BLM, respectively. However, the risk of osteosarcoma in Werner and Bloom syndromes is lower with b 10% of affected individuals developing osteosarcoma [29,30]. Pathophysiology of human OS Human OS is a disease characterised by a lack of recurrent translocations and a complex karyotype (Fig. 1). Studies using chromosome karyotyping and, more recently, array based comparative genome hybridisation or single nucleotide polymorphism have demonstrated a range of alterations in the OS genome including copy number abnormalities, abnormal ploidy, and structural rearrangements [4,31,32]. These genomic approaches have identified several genes of potential importance in the development and progression of OS. For example, the gene regions containing the p53 regulators MDM2 and COPS3 are amplified in 10% and 50% of osteosarcomas, respectively and amplification of the MYC oncogene has been identified in close to half of osteosarcomas [33–36]. However, the complexity of the OS genome has limited the utility of these genomic approaches. In addition to that seen in OS arising in the context of hereditary retinoblastoma or Li–Fraumeni syndrome, the tumor suppressors Rb and p53 are very frequently altered in sporadic osteosarcomas. Alterations at the Rb locus are a frequently occurring event in sporadic OS, with almost 70% of osteosarcomas having at least one Rb gene alteration [16,37]. Homozygous deletions of Rb are seen in 23% of tumors but Rb point mutations are rarer occurring in only 6% of tumors [18,37]. In addition to direct targeting of Rb itself, numerous mutations that effectively disrupt the “Rb pathway” have been described in OS. Examples of these include loss of function at the INK4a/ARF locus and amplification of CDK4 that result in a functional disruption of the “Rb pathway” in the absence of direct mutation to Rb itself [38–40]. Collectively these observations from human OS suggest that targeting of the “Rb pathway” is central to the pathogenesis of OS, as it is thought to be in nearly all human cancer [41,42]. Evidence from mouse models of osteosarcoma, discussed further below, suggests that Rb inactivation accelerates osteosarcoma development but, alone, is not sufficient to cause osteosarcoma [43]. On the other hand, evidence from mouse models suggests that p53 inactivation is sufficient to initiate development of OS [43,44]. Approximately 50% of human osteosarcomas contain somatic p53 deletions or point mutations [45–48]. Approximately half of these somatic p53 alterations are associated with loss of the remaining normal p53 allele [46]. Several oncogenes have been identified as possibly playing a role in osteosarcoma including MET, FOS, insulin like growth factor 1 receptor, and HER2 [49]. Because the majority of deaths due to osteosarcoma are caused by the complications of metastatic disease, a good deal of effort has been focussed on identification of factors contributing to osteosarcoma metastasis. Factors that are associated with the presence of metastases in human osteosarcoma and/or with the development of metastases in animal models of osteosarcoma include high expression of the membrane-cytoskeleton linking protein Ezrin [50] and the chemokine receptor CXCR4 [51,52], and decreased expression of the pro-apoptotic FAS receptor [53]. As discussed below, the models utilized for these studies as with all
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Fig. 1. Karyotype from human metaphase osteosarcoma cell showing gain of 3 copies of chromosome 8, ring chromosome (labelled mar), additional material of unknown origin on the large arm of chromosome 6 and short arm of chromosome 11, and derivative whole-arm translocation 13;21.
model systems have limitations and so, it is not yet clear to what extent modulating these pathways will affect spontaneously arising osteosarcoma metastases in human patients. Animal models of osteosarcoma The development of osteosarcoma in rodent models has a long history. Initial models were either spontaneous in nature or resulted from exposure to radioactive agents. Of note among these early models was the development of OS in rats following the administration of P32-orthophosphate which resulted in a high incidence of OS [54,55]. Iterations of this inductive regimen have continued since these original reports, with a range of radioactive agents showing efficacy at inducing OS yet the relevance of these tumours to the most common types of spontaneously arising OS remains unclear as only a small proportion of human osteosarcomas are radiation induced [56]. An unexpected observation for which the human relevance is currently controversial and disputed were the observations that the long-term administration of parathyroid hormone 1-34 (PTH) in rats resulted in a high incidence of metastatic OS [57]. The significance of these observations to the clinical use of PTH is unclear but they do flag an interesting aspect of the initiation of OS that is yet to be explored. There is a significant body of literature related to the development and use of xenograft and allograft models of human or murine OS cells injected into immunocompromised mice. These xenograft and allograft models have yielded important research results but they have several significant limitations [58,59]. The nature of these models prevents the study of several biologically important processes: tumor–stroma interaction, immune responses to osteosarcoma and processes involved in spontaneous metastasis. In canines, particularly large breed canines, osteosarcoma is the most common primary tumor of bone occurring in approximately
8000 dogs per year in the United States. Canine osteosarcoma has not been as fully characterized as human osteosarcoma but the tumors appear to share some biological characteristics [60,61]. Research with canine osteosarcoma faces similar logistical limitations to research with human osteosarcoma. Coordinated multi-center cooperative efforts are required in order to collect sufficient tumor specimens and/or enrol a sufficient number of subjects on clinical trials. Nevertheless, canine models are and will be important because of the opportunity to conduct comparative oncology studies and to evaluate new therapies. Genetically engineered mouse models of human osteosarcoma With the development of gene targeting technology in the mouse, the ability to specifically alter individual genes (by loss or gain of function) became achievable, and as a result more refined genetic models became possible. The development of OS in genetically engineered murine models has increased our insight into the genetic basis of osteosarcoma. For example, clinical observations in Li– Fraumeni syndrome and studies of somatic genomic alterations in OS raised the possibility of a central role for p53 in osteosarcoma oncogenesis. Germ-line deletion of p53 in the mouse resulted in an OS incidence of 4% in homozygous p53 null mice [62] and an OS incidence of 25% in heterozygote p53 animals [63], confirming this hypothesis. From an OS perspective, these models based on germline p53 deletion were hampered by the relatively higher penetrance of other tumours (particularly lymphomas) and the relatively long latency of disease in p53−/− animals. There is an increased penetrance of OS relative to other tumours in the germ-line p53 mutant animals bearing mutations present in Li–Fraumeni and human tumours [44]. These studies and those subsequent using lineage restricted analysis of p53 deficiency or mutation, discussed further below, have reinforced the
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important role that p53 mutation plays in the initiation and maintenance of OS. As another example of this principle, transgenic animals overexpressing c-Fos develop OS at a high frequency, a result consistent with the increased expression of this gene in human OS [64,65]. However, the capacity to generate OS in a mouse does not necessarily correlate with relevance to the human disease. For example, the deletion of the neurofibramatosis 2 gene (Nf2) in mice resulted in a high frequency of OS in the Nf2 deficient animals, an observation that failed to be validated by identification of mutations in this gene in human OS [66,67]. Generation of osteo-lineage specific models with high penetrance of osteosarcoma The increasingly sophisticated application of conditional gene regulation (Cre-lox; tetracycline regulation of expression) to mouse genetics has led to dramatic improvements in our ability to generate lineage restricted and temporally controlled modulation of gene function to generate models of cancer in the mouse [68]. The most heavily utilized strategy to date to generate such models has been the coupling of conditionally targeted allele/s (“floxed”) with a second allele expressing Cre recombinase in the cell lineage of interest (osteo-lineage cells). Using this approach the gene of interest (p53 and/or pRb) can be inactivated in a temporally controlled fashion by the expression of Cre recombinase, which can be either constitutively expressed in the osteo-lineage or itself subject to a second level of temporal regulation through such approaches as tetracycline regulatory elements or Cre-ER fusion proteins that are tamoxifen regulated [69]. Faithful tractable mouse models of a number of human cancers have now been reported in a variety of tissues including breast [70], prostate [71] and hematopoiesis [72]. The recent generation of mouse models of human OS has relied on these technologies to allow for modulation of genes only in the osteoblast lineage, removing the confounding issue of phenotypes in other tissues that may arise earlier or concurrently to the development of OS. The generation and characterisation of a variety of osteoblast specific Cre expressing lines has greatly enhanced our ability to generate models of human OS [73]. The range of Cre expressing lines available to modulate gene expression in the bone lineage ranges from early multi-potential cells (Prx-Cre [74]) to early committed osteoblastic progenitors (Osx-Cre [75], Col1α1 2.3-Cre [76]) to late stages of mature osteoblastic differentiation (DMP-Cre [77], Ocn-Cre Skeletal Stem Cell (Mesenchymal)
[78]) (Fig. 2). Models of OS developed that closely approximate the human disease have all utilised one of these Cre transgenic lines to mediate gene deletion in osteoblastic cells [43,79,80]. Based on the strong clinical and experimental data linking p53 mutation to human OS, numerous groups have reported that the osteoblast restricted loss of p53 leads to the development of OS. OS arose with a penetrance ranging from 60-100% depending on the Cre transgenic utilised [43,79–81]. The first of these studies using osteoblast restricted analysis of p53 function also reported that p53 loss resulted in hyperproliferation of osteoblasts, possibly providing an insight into the initiating events leading to osteosarcoma [81]. These studies are in accord with the observations from the various germ-line deficient p53 models which all developed OS to one degree or another [44,63]. In keeping with the human genetic data, there is a strong co-operativity between loss of p53 and loss of the retinoblastoma protein (pRb), the other major human familial syndrome with a high preponderance of OS. When combined, deletion of both of these genes results in the generation of a completely penetrant OS model (summarized in Table 1). These mouse tumours develop many of the defining characteristics of human OS, both familial and sporadic, such as karyotypic complexity, gene expression signatures, histology and metastatic potential [43,82–84]. Perhaps most striking and promising aspect of these mouse models of human OS is the capacity to generate a completely penetrant metastatic model. This was achieved by the temporal control of gene deletion, through use of a tetracycline repressible Cre in the Osx-Cre transgenic mouse strain, until weaning (3–4 weeks of age). This approach yielded metastatic disease in all animals with the most common sites of disease being lung and liver. A major application of these models should be addressing both the genetics of OS metastatic spread and their use as preclinical testing grounds for therapeutic interventions targeting metastatic disease initiation and maintenance which is the major cause of OS mortality. These studies have also elucidated the dominance of mutation of p53 in the development of OS when compared directly with mutation of pRb. In contrast to prevailing literature, loss of pRb alone from osteoblasts does not generate OS and results in a relatively subtle skeletal phenotype [85–87]. OS has not been reported in osteoblast specific deficiency of pRb models by independent groups [43,79,80]. These observations suggest an absolute requirement for mutation of p53 and a co-operative role for pRb. Such a hypothesis is supported by clinical observations of radiation induced sarcomas in retinoblastoma patients, where homozygous loss of p53 was reported with retention
Alternate mesenchymal fates: muscle, chondrocytes Osteocyte
Adipogenic Fate SOST DMP1
Osteogenic / Adipogenic Precursor
Lining cell Runx2
Osterix
Alk Phos Collagen1
Osteocalcin Bone Sialoprotein
Apoptosis
PTHR1
Osteoblastic Progenitor
Preosteoblast
Proliferation
Mature Osteoblast Differentiation
2.3kB Col1α1-Cre Osx-Cre 3.6kB Col1α1-Cre Prx-Cre Fig. 2. Schematic representation of the approximate cell stage of activation of the transgenic Cre lines that have been used to generate osteosarcoma models involving the conditional deletion of p53 and pRb alleles.
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Table 1 Summary of penetrance and latency of described osteosarcoma models. Cre
Gene
OS penetrance
Metastatic disease
Median survival
Reference
MSC/progenitor
Prx-1 Col1α1-3.6 Osx1
61% OS (32% PDSTS; 3% lymphoma; 3% lymphosarcoma) 29% OS (57% PDSTS; 14% lymphoma) 60% OS (20% lymphoma; 20% fibrosarcoma) 100% OS 100% OS 100% OS (20% concurrent adipogenic tumours) 75% OS (multiple tumour types per animal—also reported neuroendocine, hibernoma and rhabdomyosarcoma) 100% OS (note: Cre activity suppressed until weaning through doxycycline administration in drinking water) 85% OS (15% PDSTS)
Yes (24%)
Osteoprogenitor
p53fl/fl p53fl/fl Rbfl/fl p53fl/fl p53fl/fl
51 weeks 19 weeks 42 weeks ~ 42 weeks ~ 40 weeks ~ 18 weeks ~ 21 weeks
Lin et al. 80 Lin et al. 80 Lengner et al. 81 Walkley et al. 43 Berman et al. 87 Walkley et al. 43 Berman et al. 87
32 weeks
Walkley et al. 43
48 weeks
Lin et al. 80
p53fl/fl Rbfl/fl
Osteoblast
Osx1
p53fl/fl Rbfl/fl
Col1α1
p53fl/fl
Yes (40%)a Yes (32%) Yes (37%)
Yes (100%)a
PDSTS—poorly differentiated soft tissue sarcoma. a Small cohort of animals.
of the remaining RB allele in OS arising following radiotherapy [88,89]. Establishing and validating the relative importance of gain or loss of function mutations will now be possible in vivo. Refinements Recently reported murine models of OS have significantly advanced the field of human OS modelling however several refinements may increase their genetic similarity to the corresponding human cancer. The currently described models all utilise a loss of function allele of p53 that is less common in human cancers than point mutant alleles which result in loss of function and potential gain of function as dominant negative alleles [44]. The generation of osteoblast specific expression of the cancer associated p53 mutations may lead to osteosarcomas that more closely approximate the human cancer. The relevance of alternate mutations which result in loss of function of p53 (ARF mutation, Mdm2 overexpression) or pRb (p16 mutation/methylation) also have not been explored. The application of in vivo shRNA to these models also bears consideration as a means to model therapeutic effect with regulatable hairpin systems that would allow effects of gene loss of function on initiation and maintenance of OS to be established [90,91]. Such approaches are also highly suitable to the identification of potential therapeutic targets as they are able to be rapidly generated compared to traditional knockout approaches and they are cost effective as analysis of multiple candidate genes is possible.
which more closely approximates the pathogenic processes involved in metastatic dissemination than is possible in orthotopic models. In addition to modelling the initiation and development of OS, these murine models also provide an opportunity to more clearly define the likely candidate tumour initiating cell of osteosarcoma [92]. By sequential targeting of gene deletion from most primitive (multipotential mesenchymal cell) to most mature osteoblastic cell (mature osteoblast to osteocyte) it is possible to determine not only which cells are capable of giving rise to OS in vivo but also to determine if the developmental stage of deletion impacts of disease latency, penetrance or behaviour. Such studies can be complemented with in vitro gene deletion after isolation of phenotypically enriched subsets of skeletal progenitor cells using viral Cre approaches. To date studies have been reported using Prx-Cre (early multipotential cells of the limb bud [80]), the Col1α1 3.6-Cre transgenic (osteoprogenitors [81]), Osx-Cre (osteoprogenitors [43,79]) and Col1a1-Cre (committed osteoblast [80]). What is most striking about these data sets is that irrespective of the stage of development at which Cre becomes active in the respective transgenic lines the latency of OS is essentially the same when comparing either p53 deficiency alone or in combination with pRb. The use of Cre lines expressed in more primitive cells (Prx) does however lead to the development of tumours of other mesenchymal lineages at higher frequency. Informed by the genetics of the mouse models of OS it will be possible to formally demonstrate the most likely cancer initiating population in OS in vivo, a study which would pose numerous problems in either the canine or human xenograft system.
Applications of murine models The development of the murine models has been largely informed by the clinical genetics of human OS. The development of these models now allows for their application in a more detailed understanding of the genetics of the initiation and maintenance of osteosarcoma and the development of metastasis. The isolation and ready establishment of cell lines from both primary and paired metastatic lesions allows for the large scale screening of primary OS cells and metastatic cells using either chemical library screening or shRNA/siRNA libraries. These approaches offer the opportunity to more rapidly identify new candidate therapeutics as well as genetic interactions that may be able to be therapeutically exploited. Once identified any putative hits from such screening efforts are able to be tested in vivo in the established models of OS. The use of in vivo imaging modalities (PET/μCT) will allow for studies designed analogous to human clinical trials. A second opportunity arising from the establishment and validation of these models relates to the capacity to generate a completely penetrant metastatic disease model. Metastatic disease represents the major clinical challenge in OS and these models now allow for an active analysis of the process and genetics of OS as well as the possibility of in vivo screening against spontaneously arising OS
Conclusions The generation of tractable animal models of human disease offers many opportunities to significantly advance our understanding of the biology and genetics of human cancer. The increasing sophistication with which gene expression can be modulated in the mouse, both positively and negatively in addition to temporally, allows for the generation of more faithful models, which can recapitulate all aspects of the disease process from OS initiation and establishment to invasion and dissemination to distant sites. Modern conditionally engineered murine osteosarcoma models, when coupled with efforts utilizing human samples such as those of the NCI Sponsored Pediatric Preclinical Testing Program (PPTP) [59] and comparative oncology approaches using canine disease, have a great potential to facilitate clinically relevant biologic advances and pre-clinical drug evaluation allowing rational selection of drugs for clinical trials. Osteosarcoma is relatively uncommon and it occurs most often in adolescents and young adults, a patient population that is less likely to enrol on clinical trials [7]. Due to these factors, multi-institutional phase III trials in osteosarcoma are likely to require at least 4 years of patient enrolment, making rational selection of drugs for study in phase III
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trials essential if the outcomes of osteosarcoma are to be substantively improved [8]. Acknowledgments We thank Dr Tony Mutsaers for critical reading and comment. Work in C.R.W's laboratory is supported by grants from the NHMRC of Australia. C.R.W. was a Special Fellow of the Leukemia & Lymphoma Society, an NHMRC Career Development Award holder and is the Leukaemia Foundation Phillip Desbrow Senior Research Fellow. References [1] Parkin DM, Stiller CA, Nectoux J. International variations in the incidence of childhood bone tumours. Int J Cancer 1993;53:371–6. [2] Mirabello L, Troisi RJ, Savage SA. International osteosarcoma incidence patterns in children and adolescents, middle ages and elderly persons. Int J Cancer 2009;125: 229–34. [3] Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer 2009;115:1531–43. [4] Sandberg AA, Bridge JA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: osteosarcoma and related tumors. Cancer Genet Cytogenet 2003;145:1–30. [5] Meyers PA, Schwartz CL, Krailo M, Kleinerman ES, Betcher D, Bernstein ML, et al. Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/ or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol 2005;23:2004–11. [6] A.E. Bleyer. Cancer Epidemiology in Older Adolescents and Young Adults 15 to 29 Years of Age, Including SEER Incidence and Survival: 1975–2000. In: National Cancer Institute N, editor. Bethesda: National Cancer Institute, NIH; 2006. p. Pub. No. 06–5767. [7] Shochat SJ, Fremgen AM, Murphy SB, Hutchison C, Donaldson SS, Haase GM, et al. Childhood cancer: patterns of protocol participation in a national survey. CA Cancer J Clin 2001;51:119–30. [8] Meyers PA, Schwartz CL, Krailo MD, Healey JH, Bernstein ML, Betcher D, et al. Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival—a report from the Children's Oncology Group. J Clin Oncol 2008;26:633–8. [9] Goorin AM, Harris MB, Bernstein M, Ferguson W, Devidas M, Siegal GP, et al. Phase II/III trial of etoposide and high-dose ifosfamide in newly diagnosed metastatic osteosarcoma: a pediatric oncology group trial. J Clin Oncol 2002;20:426–33. [10] Gorlick R, Anderson P, Andrulis I, Arndt C, Beardsley GP, Bernstein M, et al. Biology of childhood osteogenic sarcoma and potential targets for therapeutic development: meeting summary. Clin Cancer Res 2003;9:5442–53. [11] Li FP, Fraumeni Jr JF. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med 1969;71:747–52. [12] Frebourg T, Barbier N, Yan YX, Garber JE, Dreyfus M, Fraumeni Jr J, et al. Germ-line p53 mutations in 15 families with Li–Fraumeni syndrome. Am J Hum Genet 1995;56:608–15. [13] Frebourg T, Friend SH. Cancer risks from germline p53 mutations. J Clin Invest 1992;90:1637–41. [14] Frebourg T, Kassel J, Lam KT, Gryka MA, Barbier N, Andersen TI, et al. Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein. Proc Natl Acad Sci USA 1992;89:6413–7. [15] Porter DE, Holden ST, Steel CM, Cohen BB, Wallace MR, Reid R. A significant proportion of patients with osteosarcoma may belong to Li–Fraumeni cancer families. J Bone Joint Surg Br 1992;74:883–6. [16] Hansen MF, Koufos A, Gallie BL, Phillips RA, Fodstad O, Brogger A, et al. Osteosarcoma and retinoblastoma: a shared chromosomal mechanism revealing recessive predisposition. Proc Natl Acad Sci USA 1985;82:6216–20. [17] Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986;323:643–6. [18] Friend SH, Horowitz JM, Gerber MR, Wang XF, Bogenmann E, Li FP, et al. Deletions of a DNA sequence in retinoblastomas and mesenchymal tumors: organization of the sequence and its encoded protein. Proc Natl Acad Sci USA 1987;84:9059–63. [19] Hansen MF, Cavenee WK. Retinoblastoma and osteosarcoma: the prototypic cancer family. Acta Paediatr Jpn 1987;29:526–33. [20] Dick DC, Morley WN, Watson JT. Rothmund–Thomson syndrome and osteogenic sarcoma. Clin Exp Dermatol 1982;7:119–23. [21] Judge MR, Kilby A, Harper JI. Rothmund–Thomson syndrome and osteosarcoma. Br J Dermatol 1993;129:723–5. [22] Kitao S, Shimamoto A, Goto M, Miller RW, Smithson WA, Lindor NM, et al. Mutations in RECQL4 cause a subset of cases of Rothmund–Thomson syndrome. Nat Genet 1999;22:82–4. [23] Nishijo K, Nakayama T, Aoyama T, Okamoto T, Ishibe T, Yasura K, et al. Mutation analysis of the RECQL4 gene in sporadic osteosarcomas. Int J Cancer 2004;111: 367–72. [24] Malkin D, Li FP, Strong LC, Fraumeni Jr JF, Nelson CE, Kim DH, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990;250:1233–8.
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Contents lists available at ScienceDirect
Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Review
Modeling human osteosarcoma in the mouse: From bedside to bench Katherine A. Janeway a,⁎, Carl R. Walkley b,⁎ a
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, 44 Binney St, Boston, MA 02115, USA b St. Vincent's Institute of Medical Research & Department of Medicine, University of Melbourne, 9 Princes St, Fitzroy, Victoria 3065, Australia
a r t i c l e
i n f o
Article history: Received 7 June 2010 Revised 28 July 2010 Accepted 30 July 2010 Available online 6 August 2010 Edited by: T. Jack Martin Keywords: Osteosarcoma Mouse model Bone biology Cancer
a b s t r a c t Osteosarcoma (OS) is the most common primary tumour of bone, occurring predominantly in the second decade of life. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized (non-metastatic) disease approaching 70%. At presentation approximately 20% of patients have metastases and almost all patients with recurrent OS have metastatic disease and cure rates for patients with metastatic or recurrent disease remain poor (b 20% survival). Over the past 20 years, considerable progress has been made in the understanding of OS pathogenesis, yet these insights have not translated into substantial therapeutic advances and clinical outcomes. Further progress is essential in order to develop molecularly based therapies that target both primary lesions as well as metastatic disease. The increasing sophistication with which gene expression can be modulated in the mouse, both positively and negatively in addition to temporally, has allowed for the recent generation of more faithful OS models than have previously been available. These murine OS models can recapitulate all aspects of the disease process, from initiation and establishment to invasion and dissemination to distant sites. The development and utilisation of murine models that faithfully recapitulate human osteosarcoma, complementing existing approaches using human and canine disease, holds significant promise in furthering our understanding of the genetic basis of the disease and, more critically, in advancing pre-clinical studies aimed at the rational development and trialing of new therapeutic approaches. © 2010 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges in the clinical study of human osteosarcoma . . . . . . . . . . . . . Human osteosarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic susceptibility to OS in humans. . . . . . . . . . . . . . . . . . . . Pathophysiology of human OS . . . . . . . . . . . . . . . . . . . . . . . . Animal models of osteosarcoma . . . . . . . . . . . . . . . . . . . . . . . . . Genetically engineered mouse models of human osteosarcoma . . . . . . . . . . Generation of osteo-lineage specific models with high penetrance of osteosarcoma . Refinements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of murine models . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction
⁎ Corresponding authors. Fax: +61 3 9416 2676. E-mail addresses: [email protected] (K.A. Janeway), [email protected] (C.R. Walkley). 8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2010.07.028
Osteosarcoma (OS) is the most common primary tumour of bone, occurring predominantly in the second decade of life. It is the most frequently occurring paediatric non-hematological tumour of bone and the 5th most prevalent malignancy of adolescence [1]. OS is
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notable for its locally aggressive behaviour and early metastasis formation. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized (non-metastatic) disease approaching 70%, but at the cost of considerable therapy-related morbidity [2,3]. At presentation approximately 20% of patients have metastases and almost all patients with recurrent OS have metastatic disease. Cure rates for patients with metastatic or recurrent disease remain poor (b20% survival) [4,5]. Over the past 20 years, considerable progress has been made in the understanding of OS pathogenesis. These insights have not yet been translated into substantial therapeutic advances and clinical benefit. Further progress is essential in order to develop molecularly based therapies that target both primary lesions as well as metastatic disease. The development and utilisation of animal models that faithfully recapitulate human osteosarcoma holds significant promise in furthering our understanding of the genetic basis of the disease and, more critically, in advancing pre-clinical studies aimed at the trialing and rational development of new therapeutic approaches. Challenges in the clinical study of human osteosarcoma Despite being the most common primary tumor of bone, osteosarcoma is relatively rare with approximately 600 patients developing osteosarcoma each year in the United States [6]. Osteosarcoma occurs most often in adolescents and young adults, a patient population that is less likely to enrol on clinical trials [7]. Due to these factors, national, multi-institutional phase III trials in osteosarcoma typically require at least 4 years of patient enrolment, making rational selection of drugs for study in phase III trials essential [8]. However, selecting drugs to study in phase III trials in osteosarcoma is complicated by several factors. Because of the bony matrix, even when exposed to highly effective therapy, osteosarcoma may not demonstrate a radiographic response [9]. Due to the complexity of osteosarcoma biology, research has, so far, led to only a limited number of clinically relevant biologic insights [10]. In addition, standard of care for many recurrent osteosarcomas is surgical resection, complicating the conduct of phase II clinical trials in osteosarcoma. Due to these and other factors, there has been only one significant therapeutic advance in osteosarcoma in the past 20 years, the addition of Liposomal Muramyl-Tripeptide Phosphatidyl Ethanolamine (L-MTP-PE) to standard upfront chemotherapy resulting in a modest improvement in overall survival [8]. Given the considerable challenges inherent in clinical drug development for osteosarcoma, novel approaches to drug and drug target discovery, with particular attention to drug development for metastatic osteosarcoma, are needed. Human osteosarcoma Genetic susceptibility to OS in humans Familial cancer syndromes, whilst rare, have provided important insight in to the underlying genetics of human disease. With respect to OS, three cancer predisposition syndromes stand out as significantly increasing the incidence of OS. These are Li–Fraumeni syndrome [11– 15], hereditary retinoblastoma [16–19] and Rothmund–Thomson and other syndromes associated with mutations in the RECQL DNA helicases [20–23]. Li–Fraumeni syndrome is due to germ-line heterozygous loss of function mutations in p53 [24]. Li–Fraumeni syndrome predisposes affected individuals to a wide variety of cancers, most commonly breast, brain tumors, leukemia, and bone and soft tissue sarcomas. Approximately 10% of malignancies occurring in patients with Li– Fraumeni syndrome are osteosarcoma [25]. Individuals with hereditary retinoblastoma carry a germline deletion or inactivating mutation in one allele of the retinoblastoma 1 gene (Rb) and develop
retinoblastoma, a malignant tumor of the retina, with a penetrance of 90%. Approximately 50% of the malignancies other than retinoblastoma developing in individuals with hereditary retinoblastoma are osteosarcomas [26]. Rothmund–Thomson syndrome is an autosomal recessive cancer predisposition syndrome caused by germline mutations of RECQL4, a DNA helicase [27,28]. This syndrome is characterized by sparse hair growth, skin rash, skeletal dysplasia and an increased risk of osteosarcoma with 30% of patients with Rothmund– Thomson syndrome developing osteosarcoma. Werner and Bloom syndromes are related to Rothmund–Thomson in that they are caused by mutations in RECQ DNA helicases, WRN and BLM, respectively. However, the risk of osteosarcoma in Werner and Bloom syndromes is lower with b 10% of affected individuals developing osteosarcoma [29,30]. Pathophysiology of human OS Human OS is a disease characterised by a lack of recurrent translocations and a complex karyotype (Fig. 1). Studies using chromosome karyotyping and, more recently, array based comparative genome hybridisation or single nucleotide polymorphism have demonstrated a range of alterations in the OS genome including copy number abnormalities, abnormal ploidy, and structural rearrangements [4,31,32]. These genomic approaches have identified several genes of potential importance in the development and progression of OS. For example, the gene regions containing the p53 regulators MDM2 and COPS3 are amplified in 10% and 50% of osteosarcomas, respectively and amplification of the MYC oncogene has been identified in close to half of osteosarcomas [33–36]. However, the complexity of the OS genome has limited the utility of these genomic approaches. In addition to that seen in OS arising in the context of hereditary retinoblastoma or Li–Fraumeni syndrome, the tumor suppressors Rb and p53 are very frequently altered in sporadic osteosarcomas. Alterations at the Rb locus are a frequently occurring event in sporadic OS, with almost 70% of osteosarcomas having at least one Rb gene alteration [16,37]. Homozygous deletions of Rb are seen in 23% of tumors but Rb point mutations are rarer occurring in only 6% of tumors [18,37]. In addition to direct targeting of Rb itself, numerous mutations that effectively disrupt the “Rb pathway” have been described in OS. Examples of these include loss of function at the INK4a/ARF locus and amplification of CDK4 that result in a functional disruption of the “Rb pathway” in the absence of direct mutation to Rb itself [38–40]. Collectively these observations from human OS suggest that targeting of the “Rb pathway” is central to the pathogenesis of OS, as it is thought to be in nearly all human cancer [41,42]. Evidence from mouse models of osteosarcoma, discussed further below, suggests that Rb inactivation accelerates osteosarcoma development but, alone, is not sufficient to cause osteosarcoma [43]. On the other hand, evidence from mouse models suggests that p53 inactivation is sufficient to initiate development of OS [43,44]. Approximately 50% of human osteosarcomas contain somatic p53 deletions or point mutations [45–48]. Approximately half of these somatic p53 alterations are associated with loss of the remaining normal p53 allele [46]. Several oncogenes have been identified as possibly playing a role in osteosarcoma including MET, FOS, insulin like growth factor 1 receptor, and HER2 [49]. Because the majority of deaths due to osteosarcoma are caused by the complications of metastatic disease, a good deal of effort has been focussed on identification of factors contributing to osteosarcoma metastasis. Factors that are associated with the presence of metastases in human osteosarcoma and/or with the development of metastases in animal models of osteosarcoma include high expression of the membrane-cytoskeleton linking protein Ezrin [50] and the chemokine receptor CXCR4 [51,52], and decreased expression of the pro-apoptotic FAS receptor [53]. As discussed below, the models utilized for these studies as with all
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Fig. 1. Karyotype from human metaphase osteosarcoma cell showing gain of 3 copies of chromosome 8, ring chromosome (labelled mar), additional material of unknown origin on the large arm of chromosome 6 and short arm of chromosome 11, and derivative whole-arm translocation 13;21.
model systems have limitations and so, it is not yet clear to what extent modulating these pathways will affect spontaneously arising osteosarcoma metastases in human patients. Animal models of osteosarcoma The development of osteosarcoma in rodent models has a long history. Initial models were either spontaneous in nature or resulted from exposure to radioactive agents. Of note among these early models was the development of OS in rats following the administration of P32-orthophosphate which resulted in a high incidence of OS [54,55]. Iterations of this inductive regimen have continued since these original reports, with a range of radioactive agents showing efficacy at inducing OS yet the relevance of these tumours to the most common types of spontaneously arising OS remains unclear as only a small proportion of human osteosarcomas are radiation induced [56]. An unexpected observation for which the human relevance is currently controversial and disputed were the observations that the long-term administration of parathyroid hormone 1-34 (PTH) in rats resulted in a high incidence of metastatic OS [57]. The significance of these observations to the clinical use of PTH is unclear but they do flag an interesting aspect of the initiation of OS that is yet to be explored. There is a significant body of literature related to the development and use of xenograft and allograft models of human or murine OS cells injected into immunocompromised mice. These xenograft and allograft models have yielded important research results but they have several significant limitations [58,59]. The nature of these models prevents the study of several biologically important processes: tumor–stroma interaction, immune responses to osteosarcoma and processes involved in spontaneous metastasis. In canines, particularly large breed canines, osteosarcoma is the most common primary tumor of bone occurring in approximately
8000 dogs per year in the United States. Canine osteosarcoma has not been as fully characterized as human osteosarcoma but the tumors appear to share some biological characteristics [60,61]. Research with canine osteosarcoma faces similar logistical limitations to research with human osteosarcoma. Coordinated multi-center cooperative efforts are required in order to collect sufficient tumor specimens and/or enrol a sufficient number of subjects on clinical trials. Nevertheless, canine models are and will be important because of the opportunity to conduct comparative oncology studies and to evaluate new therapies. Genetically engineered mouse models of human osteosarcoma With the development of gene targeting technology in the mouse, the ability to specifically alter individual genes (by loss or gain of function) became achievable, and as a result more refined genetic models became possible. The development of OS in genetically engineered murine models has increased our insight into the genetic basis of osteosarcoma. For example, clinical observations in Li– Fraumeni syndrome and studies of somatic genomic alterations in OS raised the possibility of a central role for p53 in osteosarcoma oncogenesis. Germ-line deletion of p53 in the mouse resulted in an OS incidence of 4% in homozygous p53 null mice [62] and an OS incidence of 25% in heterozygote p53 animals [63], confirming this hypothesis. From an OS perspective, these models based on germline p53 deletion were hampered by the relatively higher penetrance of other tumours (particularly lymphomas) and the relatively long latency of disease in p53−/− animals. There is an increased penetrance of OS relative to other tumours in the germ-line p53 mutant animals bearing mutations present in Li–Fraumeni and human tumours [44]. These studies and those subsequent using lineage restricted analysis of p53 deficiency or mutation, discussed further below, have reinforced the
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important role that p53 mutation plays in the initiation and maintenance of OS. As another example of this principle, transgenic animals overexpressing c-Fos develop OS at a high frequency, a result consistent with the increased expression of this gene in human OS [64,65]. However, the capacity to generate OS in a mouse does not necessarily correlate with relevance to the human disease. For example, the deletion of the neurofibramatosis 2 gene (Nf2) in mice resulted in a high frequency of OS in the Nf2 deficient animals, an observation that failed to be validated by identification of mutations in this gene in human OS [66,67]. Generation of osteo-lineage specific models with high penetrance of osteosarcoma The increasingly sophisticated application of conditional gene regulation (Cre-lox; tetracycline regulation of expression) to mouse genetics has led to dramatic improvements in our ability to generate lineage restricted and temporally controlled modulation of gene function to generate models of cancer in the mouse [68]. The most heavily utilized strategy to date to generate such models has been the coupling of conditionally targeted allele/s (“floxed”) with a second allele expressing Cre recombinase in the cell lineage of interest (osteo-lineage cells). Using this approach the gene of interest (p53 and/or pRb) can be inactivated in a temporally controlled fashion by the expression of Cre recombinase, which can be either constitutively expressed in the osteo-lineage or itself subject to a second level of temporal regulation through such approaches as tetracycline regulatory elements or Cre-ER fusion proteins that are tamoxifen regulated [69]. Faithful tractable mouse models of a number of human cancers have now been reported in a variety of tissues including breast [70], prostate [71] and hematopoiesis [72]. The recent generation of mouse models of human OS has relied on these technologies to allow for modulation of genes only in the osteoblast lineage, removing the confounding issue of phenotypes in other tissues that may arise earlier or concurrently to the development of OS. The generation and characterisation of a variety of osteoblast specific Cre expressing lines has greatly enhanced our ability to generate models of human OS [73]. The range of Cre expressing lines available to modulate gene expression in the bone lineage ranges from early multi-potential cells (Prx-Cre [74]) to early committed osteoblastic progenitors (Osx-Cre [75], Col1α1 2.3-Cre [76]) to late stages of mature osteoblastic differentiation (DMP-Cre [77], Ocn-Cre Skeletal Stem Cell (Mesenchymal)
[78]) (Fig. 2). Models of OS developed that closely approximate the human disease have all utilised one of these Cre transgenic lines to mediate gene deletion in osteoblastic cells [43,79,80]. Based on the strong clinical and experimental data linking p53 mutation to human OS, numerous groups have reported that the osteoblast restricted loss of p53 leads to the development of OS. OS arose with a penetrance ranging from 60-100% depending on the Cre transgenic utilised [43,79–81]. The first of these studies using osteoblast restricted analysis of p53 function also reported that p53 loss resulted in hyperproliferation of osteoblasts, possibly providing an insight into the initiating events leading to osteosarcoma [81]. These studies are in accord with the observations from the various germ-line deficient p53 models which all developed OS to one degree or another [44,63]. In keeping with the human genetic data, there is a strong co-operativity between loss of p53 and loss of the retinoblastoma protein (pRb), the other major human familial syndrome with a high preponderance of OS. When combined, deletion of both of these genes results in the generation of a completely penetrant OS model (summarized in Table 1). These mouse tumours develop many of the defining characteristics of human OS, both familial and sporadic, such as karyotypic complexity, gene expression signatures, histology and metastatic potential [43,82–84]. Perhaps most striking and promising aspect of these mouse models of human OS is the capacity to generate a completely penetrant metastatic model. This was achieved by the temporal control of gene deletion, through use of a tetracycline repressible Cre in the Osx-Cre transgenic mouse strain, until weaning (3–4 weeks of age). This approach yielded metastatic disease in all animals with the most common sites of disease being lung and liver. A major application of these models should be addressing both the genetics of OS metastatic spread and their use as preclinical testing grounds for therapeutic interventions targeting metastatic disease initiation and maintenance which is the major cause of OS mortality. These studies have also elucidated the dominance of mutation of p53 in the development of OS when compared directly with mutation of pRb. In contrast to prevailing literature, loss of pRb alone from osteoblasts does not generate OS and results in a relatively subtle skeletal phenotype [85–87]. OS has not been reported in osteoblast specific deficiency of pRb models by independent groups [43,79,80]. These observations suggest an absolute requirement for mutation of p53 and a co-operative role for pRb. Such a hypothesis is supported by clinical observations of radiation induced sarcomas in retinoblastoma patients, where homozygous loss of p53 was reported with retention
Alternate mesenchymal fates: muscle, chondrocytes Osteocyte
Adipogenic Fate SOST DMP1
Osteogenic / Adipogenic Precursor
Lining cell Runx2
Osterix
Alk Phos Collagen1
Osteocalcin Bone Sialoprotein
Apoptosis
PTHR1
Osteoblastic Progenitor
Preosteoblast
Proliferation
Mature Osteoblast Differentiation
2.3kB Col1α1-Cre Osx-Cre 3.6kB Col1α1-Cre Prx-Cre Fig. 2. Schematic representation of the approximate cell stage of activation of the transgenic Cre lines that have been used to generate osteosarcoma models involving the conditional deletion of p53 and pRb alleles.
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863
Table 1 Summary of penetrance and latency of described osteosarcoma models. Cre
Gene
OS penetrance
Metastatic disease
Median survival
Reference
MSC/progenitor
Prx-1 Col1α1-3.6 Osx1
61% OS (32% PDSTS; 3% lymphoma; 3% lymphosarcoma) 29% OS (57% PDSTS; 14% lymphoma) 60% OS (20% lymphoma; 20% fibrosarcoma) 100% OS 100% OS 100% OS (20% concurrent adipogenic tumours) 75% OS (multiple tumour types per animal—also reported neuroendocine, hibernoma and rhabdomyosarcoma) 100% OS (note: Cre activity suppressed until weaning through doxycycline administration in drinking water) 85% OS (15% PDSTS)
Yes (24%)
Osteoprogenitor
p53fl/fl p53fl/fl Rbfl/fl p53fl/fl p53fl/fl
51 weeks 19 weeks 42 weeks ~ 42 weeks ~ 40 weeks ~ 18 weeks ~ 21 weeks
Lin et al. 80 Lin et al. 80 Lengner et al. 81 Walkley et al. 43 Berman et al. 87 Walkley et al. 43 Berman et al. 87
32 weeks
Walkley et al. 43
48 weeks
Lin et al. 80
p53fl/fl Rbfl/fl
Osteoblast
Osx1
p53fl/fl Rbfl/fl
Col1α1
p53fl/fl
Yes (40%)a Yes (32%) Yes (37%)
Yes (100%)a
PDSTS—poorly differentiated soft tissue sarcoma. a Small cohort of animals.
of the remaining RB allele in OS arising following radiotherapy [88,89]. Establishing and validating the relative importance of gain or loss of function mutations will now be possible in vivo. Refinements Recently reported murine models of OS have significantly advanced the field of human OS modelling however several refinements may increase their genetic similarity to the corresponding human cancer. The currently described models all utilise a loss of function allele of p53 that is less common in human cancers than point mutant alleles which result in loss of function and potential gain of function as dominant negative alleles [44]. The generation of osteoblast specific expression of the cancer associated p53 mutations may lead to osteosarcomas that more closely approximate the human cancer. The relevance of alternate mutations which result in loss of function of p53 (ARF mutation, Mdm2 overexpression) or pRb (p16 mutation/methylation) also have not been explored. The application of in vivo shRNA to these models also bears consideration as a means to model therapeutic effect with regulatable hairpin systems that would allow effects of gene loss of function on initiation and maintenance of OS to be established [90,91]. Such approaches are also highly suitable to the identification of potential therapeutic targets as they are able to be rapidly generated compared to traditional knockout approaches and they are cost effective as analysis of multiple candidate genes is possible.
which more closely approximates the pathogenic processes involved in metastatic dissemination than is possible in orthotopic models. In addition to modelling the initiation and development of OS, these murine models also provide an opportunity to more clearly define the likely candidate tumour initiating cell of osteosarcoma [92]. By sequential targeting of gene deletion from most primitive (multipotential mesenchymal cell) to most mature osteoblastic cell (mature osteoblast to osteocyte) it is possible to determine not only which cells are capable of giving rise to OS in vivo but also to determine if the developmental stage of deletion impacts of disease latency, penetrance or behaviour. Such studies can be complemented with in vitro gene deletion after isolation of phenotypically enriched subsets of skeletal progenitor cells using viral Cre approaches. To date studies have been reported using Prx-Cre (early multipotential cells of the limb bud [80]), the Col1α1 3.6-Cre transgenic (osteoprogenitors [81]), Osx-Cre (osteoprogenitors [43,79]) and Col1a1-Cre (committed osteoblast [80]). What is most striking about these data sets is that irrespective of the stage of development at which Cre becomes active in the respective transgenic lines the latency of OS is essentially the same when comparing either p53 deficiency alone or in combination with pRb. The use of Cre lines expressed in more primitive cells (Prx) does however lead to the development of tumours of other mesenchymal lineages at higher frequency. Informed by the genetics of the mouse models of OS it will be possible to formally demonstrate the most likely cancer initiating population in OS in vivo, a study which would pose numerous problems in either the canine or human xenograft system.
Applications of murine models The development of the murine models has been largely informed by the clinical genetics of human OS. The development of these models now allows for their application in a more detailed understanding of the genetics of the initiation and maintenance of osteosarcoma and the development of metastasis. The isolation and ready establishment of cell lines from both primary and paired metastatic lesions allows for the large scale screening of primary OS cells and metastatic cells using either chemical library screening or shRNA/siRNA libraries. These approaches offer the opportunity to more rapidly identify new candidate therapeutics as well as genetic interactions that may be able to be therapeutically exploited. Once identified any putative hits from such screening efforts are able to be tested in vivo in the established models of OS. The use of in vivo imaging modalities (PET/μCT) will allow for studies designed analogous to human clinical trials. A second opportunity arising from the establishment and validation of these models relates to the capacity to generate a completely penetrant metastatic disease model. Metastatic disease represents the major clinical challenge in OS and these models now allow for an active analysis of the process and genetics of OS as well as the possibility of in vivo screening against spontaneously arising OS
Conclusions The generation of tractable animal models of human disease offers many opportunities to significantly advance our understanding of the biology and genetics of human cancer. The increasing sophistication with which gene expression can be modulated in the mouse, both positively and negatively in addition to temporally, allows for the generation of more faithful models, which can recapitulate all aspects of the disease process from OS initiation and establishment to invasion and dissemination to distant sites. Modern conditionally engineered murine osteosarcoma models, when coupled with efforts utilizing human samples such as those of the NCI Sponsored Pediatric Preclinical Testing Program (PPTP) [59] and comparative oncology approaches using canine disease, have a great potential to facilitate clinically relevant biologic advances and pre-clinical drug evaluation allowing rational selection of drugs for clinical trials. Osteosarcoma is relatively uncommon and it occurs most often in adolescents and young adults, a patient population that is less likely to enrol on clinical trials [7]. Due to these factors, multi-institutional phase III trials in osteosarcoma are likely to require at least 4 years of patient enrolment, making rational selection of drugs for study in phase III
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trials essential if the outcomes of osteosarcoma are to be substantively improved [8]. Acknowledgments We thank Dr Tony Mutsaers for critical reading and comment. Work in C.R.W's laboratory is supported by grants from the NHMRC of Australia. C.R.W. was a Special Fellow of the Leukemia & Lymphoma Society, an NHMRC Career Development Award holder and is the Leukaemia Foundation Phillip Desbrow Senior Research Fellow. References [1] Parkin DM, Stiller CA, Nectoux J. International variations in the incidence of childhood bone tumours. Int J Cancer 1993;53:371–6. [2] Mirabello L, Troisi RJ, Savage SA. International osteosarcoma incidence patterns in children and adolescents, middle ages and elderly persons. Int J Cancer 2009;125: 229–34. [3] Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer 2009;115:1531–43. [4] Sandberg AA, Bridge JA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: osteosarcoma and related tumors. Cancer Genet Cytogenet 2003;145:1–30. [5] Meyers PA, Schwartz CL, Krailo M, Kleinerman ES, Betcher D, Bernstein ML, et al. Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/ or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol 2005;23:2004–11. [6] A.E. Bleyer. Cancer Epidemiology in Older Adolescents and Young Adults 15 to 29 Years of Age, Including SEER Incidence and Survival: 1975–2000. In: National Cancer Institute N, editor. Bethesda: National Cancer Institute, NIH; 2006. p. Pub. No. 06–5767. [7] Shochat SJ, Fremgen AM, Murphy SB, Hutchison C, Donaldson SS, Haase GM, et al. Childhood cancer: patterns of protocol participation in a national survey. CA Cancer J Clin 2001;51:119–30. [8] Meyers PA, Schwartz CL, Krailo MD, Healey JH, Bernstein ML, Betcher D, et al. Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival—a report from the Children's Oncology Group. J Clin Oncol 2008;26:633–8. [9] Goorin AM, Harris MB, Bernstein M, Ferguson W, Devidas M, Siegal GP, et al. Phase II/III trial of etoposide and high-dose ifosfamide in newly diagnosed metastatic osteosarcoma: a pediatric oncology group trial. J Clin Oncol 2002;20:426–33. [10] Gorlick R, Anderson P, Andrulis I, Arndt C, Beardsley GP, Bernstein M, et al. Biology of childhood osteogenic sarcoma and potential targets for therapeutic development: meeting summary. Clin Cancer Res 2003;9:5442–53. [11] Li FP, Fraumeni Jr JF. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med 1969;71:747–52. [12] Frebourg T, Barbier N, Yan YX, Garber JE, Dreyfus M, Fraumeni Jr J, et al. Germ-line p53 mutations in 15 families with Li–Fraumeni syndrome. Am J Hum Genet 1995;56:608–15. [13] Frebourg T, Friend SH. Cancer risks from germline p53 mutations. J Clin Invest 1992;90:1637–41. [14] Frebourg T, Kassel J, Lam KT, Gryka MA, Barbier N, Andersen TI, et al. Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein. Proc Natl Acad Sci USA 1992;89:6413–7. [15] Porter DE, Holden ST, Steel CM, Cohen BB, Wallace MR, Reid R. A significant proportion of patients with osteosarcoma may belong to Li–Fraumeni cancer families. J Bone Joint Surg Br 1992;74:883–6. [16] Hansen MF, Koufos A, Gallie BL, Phillips RA, Fodstad O, Brogger A, et al. Osteosarcoma and retinoblastoma: a shared chromosomal mechanism revealing recessive predisposition. Proc Natl Acad Sci USA 1985;82:6216–20. [17] Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986;323:643–6. [18] Friend SH, Horowitz JM, Gerber MR, Wang XF, Bogenmann E, Li FP, et al. Deletions of a DNA sequence in retinoblastomas and mesenchymal tumors: organization of the sequence and its encoded protein. Proc Natl Acad Sci USA 1987;84:9059–63. [19] Hansen MF, Cavenee WK. Retinoblastoma and osteosarcoma: the prototypic cancer family. Acta Paediatr Jpn 1987;29:526–33. [20] Dick DC, Morley WN, Watson JT. Rothmund–Thomson syndrome and osteogenic sarcoma. Clin Exp Dermatol 1982;7:119–23. [21] Judge MR, Kilby A, Harper JI. Rothmund–Thomson syndrome and osteosarcoma. Br J Dermatol 1993;129:723–5. [22] Kitao S, Shimamoto A, Goto M, Miller RW, Smithson WA, Lindor NM, et al. Mutations in RECQL4 cause a subset of cases of Rothmund–Thomson syndrome. Nat Genet 1999;22:82–4. [23] Nishijo K, Nakayama T, Aoyama T, Okamoto T, Ishibe T, Yasura K, et al. Mutation analysis of the RECQL4 gene in sporadic osteosarcomas. Int J Cancer 2004;111: 367–72. [24] Malkin D, Li FP, Strong LC, Fraumeni Jr JF, Nelson CE, Kim DH, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990;250:1233–8.
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