Original Full Length ArticleModeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA
Highlights
► Model of osteoblastic osteosarcoma ► Highly penatrant and metastatic disease ► Modeling of distinct tumor subtypes by shRNA compared to Cre:lox
Introduction
Osteosarcoma (OS) is the most common primary tumor of bone, occurring predominantly in the second decade of life. Conventional osteosarcoma presents as three major subtypes based on histological classifications: osteoblastic, fibroblastic and chondroblastic. Of the OS subtypes osteoblastic is the most common (~ 60%) with fibroblastic and chondroblastic being approximately equally represented [1]. Despite intensive research efforts, outcomes for patients with OS have not significantly improved in the last three decades [2]. Five year survival for patients with localized disease approaches 70% but falls to between 20 and 30% for patients with recurrent disease or metastasis at diagnosis [2]. Improving outcomes for these patients will require a concerted effort utilizing a number of approaches, of which accurate murine models form a key pillar [3]. In particular the ability to model and test interventions against highly penetrant and reproducible metastatic disease would be of significant advantage.
Murine models of OS generated over past decades have utilized many approaches including radiation exposure and carcinogens [4], [5]. The recent establishment of tractable genetically defined murine models based on the genetics of human disease offers a new means to understand the molecular genetics of OS and provide for further preclinical testing. Knowledge from the familial OS predisposition syndromes Li–Fraumeni syndrome and hereditary retinoblastoma has been applied to develop models that mirror human OS [6], [7], [8]. These models have used conditional Cre:lox alleles of p53 (Trp53, Li–Fraumeni syndrome) and Rb (Rb1, hereditary retinoblastoma) and a range of osteoblastic lineage Cre transgenics. OS arising in these Cre:lox models shares cardinal features with human OS. The tumors resemble conventional human medullary OS, most closely approximating the fibroblastic/undifferentiated form [6], [7]. The development of OS models that encompass the spectrum of clinical OS subtypes (osteoblastic, chondroblastic and fibroblastic) would provide a broad pre-clinical platform to advance proposed therapies in a more precise manner [9].
Rapid improvements in the fidelity of murine models of human cancer have come about through the use of Cre:lox based approaches to elicit temporal and lineage specific gene modulations [7], [10], [11]. Recently developed transgenic, tetracycline (tet)-regulated shRNA based approaches have allowed for inhibition of endogenous gene expression in vivo [12], [13]. These have demonstrated efficacy in hematopoietic tumors but have not to date been reported in solid tumor models [13], [14]. A direct comparison of Cre:lox and shRNA approaches in modeling solid tumors is yet to be reported. One outstanding question is how comparable the effects of gene knockout are compared to gene knockdown on the tumor phenotype. The knockdown of a tumor suppressor which is highly mutated in human cancer, such as p53, may result in distinct selective pressures that are not apparent when genomic deletion is used.
Here we describe a new OS model that has utilized in vivo lineage restricted shRNA technology to bring about osteoblast specific knockdown of p53. We have also assessed the effects of concomitant deletion of Rb, known to potentiate OS formation in Cre:lox models but not itself acts as an initiating event, with shRNA mediated suppression of p53. These mice develop OS, dependent on p53 loss of function, with near 100% penetrance but at longer latency than Cre:lox-based models targeting the same genetic drivers of OS. In contrast to previous models, tumors in this model more often develop in the long bone and are highly metastatic, feature similar to human OS. Compared to Cre:lox tumors, which resemble human fibroblastic or undifferentiated OS, shRNA-driven tumors demonstrate a homogenous osteoblastic OS phenotype. As such, conditional deletion and shRNA mediated knockdown are distinctive in vivo tools and are highly complementary in efforts to establish a collection of murine models that reflect the diverse pathology of human solid tumors.
Section snippets
Materials and methods
Detailed methods can be found in the supplemental section.
Transgenic knockdown of endogenous gene expression in osteoblastic cells
To establish the efficacy of transgenic short hairpin RNA (shRNA) in a lineage-restricted manner, we made use of transgenic mice harboring a microRNA-based shRNA targeting murine p53 driven by a tet-regulated TRE promoter [13]. This allele has been extensively characterized and validated to result in an efficient knock-down of both transcript and protein levels of p53 with no reported nonspecific effects [12], [19], [20]. These TRE-p53.1224 mice were crossed with Osx-Cre mice (Figs. 1A–B) [7].
Discussion
Collectively the data demonstrate that lineage restricted shRNA against p53 is able to efficiently induce solid tumors, in this iteration osteosarcoma. We propose a model whereby use of Cre:lox alleles of p53 results in acute effect on differentiation and immortalization of a pre-osteoblastic cell, ultimately leading to a fibroblastic OS (Fig. 7B). This is consistent with previous reports where loss of p53 leads to an increased expression of both Runx2 and Osterix and an increase in
Conclusion
The variation in any human cancer can only be adequately captured experimentally through the use of more than one murine model. We and others have previously described an OS model where mice present with a phenotype most similar to fibroblastic/undifferentiated OS [6], [7]. We now report a highly penetrant model of osteoblastic OS with a high frequency of metastatic dissemination. The development of this new line provides a valuable new experimental tool and establishes a model of the most
Acknowledgments
We thank T Jacks and A McMahon for providing pRbfl/fl and Osx-Cre lines respectively; SVH BioResources Centre; N Sanders for FACS sorting; M Walia for proliferation curves; I Poulton for technical assistance; and S Orkin, N Walsh, V Sankaran and J Heierhorst for comments and discussion.
This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC; to C.W. and C.W./T.J.M.); AACR–Aflac, Inc. Career Development Award for Pediatric Cancer Research
References (50)
- et al.
Adjuvant and neoadjuvant chemotherapy for osteosarcoma of the extremities: 27 year experience at Rizzoli Institute, Italy
Eur J Cancer
(2005) A blueprint for advancing genetics-based cancer therapy
Cell
(2011)- et al.
ETV6–NTRK3 fusion oncogene initiates breast cancer from committed mammary progenitors via activation of AP1 complex
Cancer Cell
(2007) - et al.
A rapid and scalable system for studying gene function in mice using conditional RNA interference
Cell
(2011) - et al.
Erythropoietin couples erythropoiesis, B-lymphopoiesis, and bone homeostasis within the bone marrow microenvironment
Blood
(2011) - et al.
Senescence of activated stellate cells limits liver fibrosis
Cell
(2008) - et al.
Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression
Cell
(2011) - et al.
A “twist box” code of p53 inactivation: twist box:p53 interaction promotes p53 degradation
Cancer Cell
(2012) - et al.
CpG methylation inactivates the transcriptional activity of the promoter of the human p53 tumor suppressor gene
Biochem Biophys Res Commun
(1997) - et al.
Update on Wnt signaling in bone cell biology and bone disease
Gene
(2012)
Evidence for an unanticipated relationship between undifferentiated pleomorphic sarcoma and embryonal rhabdomyosarcoma
Cancer Cell
Gene expression analysis of soft tissue sarcomas: characterization and reclassification of malignant fibrous histiocytoma
Mod Pathol
A conditional mouse model of synovial sarcoma: insights into a myogenic origin
Cancer Cell
Wnt11 promotes osteoblast maturation and mineralization through R-spondin 2
J Biol Chem
Reversible tumorigenesis by MYC in hematopoietic lineages
Mol Cell
Outcome for adolescent and young adult patients with osteosarcoma: a report from the Children's Oncology Group
Cancer
Genetically engineered mouse models and human osteosarcoma
Clin Sarcoma Res
Parathyroid hormone-responsive adenylate cyclase in induced transplantable osteogenic rat sarcoma
Nature
Prkar1a is an osteosarcoma tumor suppressor that defines a molecular subclass in mice
J Clin Invest
Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage
Proc Natl Acad Sci U S A
Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease
Genes Dev
Targeted mutation of p53 and Rb in mesenchymal cells of the limb bud produces sarcomas in mice
Carcinogenesis
Maximizing mouse cancer models
Nat Rev Cancer
Tissue-specific and reversible RNA interference in transgenic mice
Nat Genet
Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi
Nat Biotechnol
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Present address: Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.