Elsevier

Bone

Volume 55, Issue 1, July 2013, Pages 166-178
Bone

Original Full Length Article
Modeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA

https://doi.org/10.1016/j.bone.2013.02.016Get rights and content

Abstract

Osteosarcoma is the most common primary cancer of bone and one that predominantly affects children and adolescents. Osteoblastic osteosarcoma represents the major subtype of this tumor, with approximately equal representation of fibroblastic and chondroblastic subtypes. We and others have previously described murine models of osteosarcoma based on osteoblast-restricted Cre:lox deletion of Trp53 (p53) and Rb1 (Rb), resulting in a phenotype most similar to fibroblastic osteosarcoma in humans. We now report a model of the most prevalent form of human osteosarcoma, the osteoblastic subtype. In contrast to other osteosarcoma models that have used Cre:lox mediated gene deletion, this model was generated through shRNA-based knockdown of p53. As is the case with the human disease the shRNA tumors most frequently present in the long bones and preferentially disseminate to the lungs; feature less consistently modeled using Cre:lox approaches. Our approach allowed direct comparison of the in vivo consequences of targeting the same genetic drivers using two different technologies, Cre:lox and shRNA. This demonstrated that the effects of Cre:lox and shRNA mediated knock-down are qualitatively different, at least in the context of osteosarcoma, and yielded distinct subtypes of osteosarcoma. Through the use of complementary genetic modification strategies we have established a model of the most common clinical subtype of osteosarcoma that was not previously represented and more fully recapitulated the clinical spectrum of this cancer.

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

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    Present address: Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.

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