Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.
Journal: 2002/January - Genes and Development
ISSN: 0890-9369
Abstract:
Adenocarcinoma of the lung is the most common form of lung cancer, but the cell of origin and the stages of progression of this tumor type are not well understood. We have developed a new model of lung adenocarcinoma in mice harboring a conditionally activatable allele of oncogenic K-ras. Here we show that the use of a recombinant adenovirus expressing Cre recombinase (AdenoCre) to induce K-ras G12D expression in the lungs of mice allows control of the timing and multiplicity of tumor initiation. Through the ability to synchronize tumor initiation in these mice, we have been able to characterize the stages of tumor progression. Of particular significance, this system has led to the identification of a new cell type contributing to the development of pulmonary adenocarcinoma.
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Genes Dev 15(24): 3243-3248

Analysis of lung tumor initiation and progression using conditional expression of oncogenic <em>K-ras</em>

Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; Dana-Farber Cancer Institute, Division of Adult Oncology, Boston, Massachusetts 02115, USA; Department of Pathology, Tufts University School of Medicine and Veterinary Medicine, Boston, Massachusetts 02111, USA
Corresponding author.
Received 2001 Sep 4; Accepted 2001 Oct 24.

Abstract

Adenocarcinoma of the lung is the most common form of lung cancer, but the cell of origin and the stages of progression of this tumor type are not well understood. We have developed a new model of lung adenocarcinoma in mice harboring a conditionally activatable allele of oncogenic K-ras. Here we show that the use of a recombinant adenovirus expressing Cre recombinase (AdenoCre) to induce K-ras G12D expression in the lungs of mice allows control of the timing and multiplicity of tumor initiation. Through the ability to synchronize tumor initiation in these mice, we have been able to characterize the stages of tumor progression. Of particular significance, this system has led to the identification of a new cell type contributing to the development of pulmonary adenocarcinoma.

Keywords: K-ras, lung cancer, mouse models, Cre-loxP
Abstract

Lung cancer is the leading cause of cancer deaths worldwide (Kerr 2001). Human lung cancers are categorized into four distinct histopathological classes: adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and small cell carcinoma. The prevalence of adenocarcinoma is increasing, and adenocarcinoma is currently the most common type of lung cancer in the United States. Unlike squamous cell carcinoma, the stages of progression have not been well described for adenocarcinoma, and little is known about the cell type of origin or the characteristics of precursor lesions of pulmonary adenocarcinoma. Patients are usually diagnosed with lung cancer because of disease symptoms or incidental chest X-ray findings. Perhaps owing to the inability to recognize premalignant lesions, patients are seldom diagnosed before their cancer has reached an advanced stage (Tuveson and Jacks 1999).

Activating mutations of the K-ras oncogene are found in one-quarter to one-half of human lung adenocarcinomas. K-Ras is a membrane-associated GTPase signaling protein that regulates proliferation, differentiation, and cell survival (Campbell et al. 1998). Missense mutations at codons 12, 13, and 61 result in decreased GTPase activity and constitutive signaling. In the mouse, K-ras mutations are found in >90% of spontaneous and chemically induced lung tumors (Malkinson 1998). In a previous effort to study spontaneous K-ras mutations in vivo, we constructed a novel mouse strain harboring a latent allele of K-ras G12D (referred to as K-rasLA) capable of spontaneous activation in vivo (Johnson et al. 2001). K-rasLA mice develop a variety of tumor types, with 100% of the mice developing multiple early onset lung tumors. K-ras mice succumb at a young age from respiratory failure caused by an overwhelming number of predominantly early-stage lung lesions. This early mortality presumably limits the capacity for tumor progression. Also, in this and other mouse lung tumor models, the asynchrony of tumor development complicates analysis of tumor initiation and progression. Finally, the penetrance and multiplicity of tumor formation have been difficult to control, as some models have incomplete penetrance, but in others the number of primary lesions far exceeds the situation in humans. An ideal model would have one or a small number of tumors that could be followed over time through different stages of tumor progression.

In an effort to improve mouse lung tumor modeling, we used a Lox–Stop–Lox K-ras conditional mouse strain (referred to as LSL-K-ras G12D), in which expression of oncogenic K-ras is controlled by a removable transcriptional termination Stop element (Tuveson et al., in prep.). Floxed Stop elements have previously been used to suppress transcription of transgenic SV40 TAg (Lakso et al. 1992) as well as various reporter genes (Mao et al. 1999). We have extended the use of conditional alleles to activate a gain-of-function mutation in a cellular oncogene. The endogenous K-ras locus is targeted in the LSL-K-ras G12D strain and, therefore, endogenous levels of oncogenic K-Ras G12D protein are expressed following removal of the Stop element. Removal of the Stop element from the LSL-K-ras G12D allele was achieved by the use of an AdenoCre, which allows control of the timing, location, and multiplicity of tumor initiation. Through the ability to synchronize tumor initiation, we have characterized the early stages of tumor progression. In addition, analysis of early-stage lesions has led to the discovery of a new cell type contributing to the development of pulmonary adenocarcinoma.

Mice were sacrificed 6 wk postinfection. Lungs were step-sectioned at 100-μm intervals and stained with H&amp;E. Those lesions readily identifiable under low-magnification were counted. The median tumor number (n) of mice is shown.

Acknowledgments

The CCA antibody was a generous gift from Anil Mukherjee from the NICHD/NIH. We thank Daniel Rines for help with deconvolution microscopy, and Ilona Linnoila and Margaret McLaughlin for helpful advice and critical reading of the manuscript. This work was funded in part by grants from the NCI. T.J. is an Associate Investigator of HHMI; D.A.T. is an HHMI Physician Postdoctoral Research Fellow.

 The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Footnotes

E-MAIL ude.tim@skcajt; FAX (617) 253-9863.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.943001.

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