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Several approaches have been taken for creating murine lung cancer models. Specific inbred strains of mice are susceptible to the development of spontaneous lung tumors (JAX MTB Tumor Frequency Grid). The most sensitive strains include A/J and SWR while others range from intermediate sensitivity (BALB/c and O20), somewhat resistant (CBA and C3H) to nearly fully resistant (DBA and C57BL/6). The susceptible strains are also sensitive to chemically-induced lung tumors, and this sensitivity has been employed as a carcinogenicity bioassay (84). A polymorphism in the second intron of K-ras, that may affect gene expression levels, is one major modifier of sensitivity to lung tumorigenesis (111).

These strain differences in tumor susceptibility have been exploited for the mapping of additional loci that confer sensitivity to lung cancer. Analysis of progeny from crosses between recombinant inbred (RI) strains derived from the sensitive A/J strain and the resistant C57BL/6J strain, suggested the existence of three pulmonary adenoma susceptiblitiy (Pas) loci (55). Pas-1 was later identified by analysis of F2 progeny from a cross between strain A/J and the C3H/He resistant strain and was mapped to the distal region of chromosome 6 (24). Linkage analysis has demonstrated K-ras to be tightly linked to the Pas-1 locus, suggesting K-ras as a candidate for Pas-1 (47). Additional Pas loci have been mapped to chromosomes 9, 17 and 19 (13, 18). Furthermore, several numerous susceptibility to lung cancer (Sluc) loci have been identified by using a multilocus mapping method to analyze F2 mice generated from recombinant congenic strains (RCS). The Sluc loci are involved in complex genetic interactions that control susceptibility to the development of lung cancer (19, 20).

Diagnosis: Keratinizing squamous carcinoma of the lung. Species: Mouse Boston reference set number: LW071A

A wide variety of chemical carcinogens can induce pulmonary adenoma and adenocarcinoma formation in mice although they vary in their potencies (for a review on spontaneous and chemically induced mouse lung tumors see (53, 84, 86)). Some well characterized tumorigenic agents include urethane, metals, aflatoxin, tobacco smoke and tobacco smoke constituents including polyaromatic hydrocarbons and nitrosamines. Of note, the only murine model of squamous cell carcinoma existing to date is a carcinogen induced model resulting from the topical administration of Nitrosobris-(2-chloroethyl) urea (NTCU) twice a week for 35-40 weeks (71). The study of chemically induced lung tumors has provided insights into the histiogenesis of murine lung tumors suggesting that murine pulmonary adenocarcinomas are derived from cells of the alveolar epithelium of the type II cell lineage, or from the bronchiolar epithelium of the Clara cell lineage (34, 70, 72, 92).

Murine lung tumors are examined both histologically and immunohistochemically in order to gain insights into the histiogenesis of the tumors. Anti-SP-C staining is commonly used to identify cells of the alveolar type II cell lineage, while anti-CC10 staining is used to identify cells of the Clara cell lineage. Anti-SPA staining has also been widely used, but is slightly less informative as both Clara cells and type II cells express SP-A. These findings have provided a basis for further investigation into the cell of origin of lung tumors. Furthermore, numerous molecular alterations have been identified in spontaneous and carcinogen induced murine lung tumors including activating mutations of the K-ras oncogene, overexpression of c-myc and decreased expression of the Rb tumor suppressor gene (12, 29, 69). Many of these findings correlate with known genetic changes in human lung tumors, suggesting an important role in the formation of pulmonary carcinomas and providing a basis for the development of transgenic models.

Diagnosis: Papillary adenocarcinoma of the lung. Species: Mouse Boston reference set number: LW003

Several transgenic lung tumor models have been created in order to direct oncogene expression to a specific subset of lung epithelial cells and to examine the role of specific oncogenes in lung tumorigenesis. Various combinations of oncogenes and cell type specific expression have resulted in murine lung tumors resembling human adenocarcinoma. Expression of SV40 T Antigen from the Clara cell specific CCSP (aka uteroglobulin, UG, or CCIO) promoter or the alveolar type II cell specific SP-C promoter develop multifocal early onset bronchioloalveolar neoplasms which progress to adenocarcinoma resulting in death by 4 months of age. (11, 79, 104). Mice constitutively overexpressing c-myc from the SP-C promoter develop pulmonary lesions ranging from bronchiolo-alveolar adenomas to adenocarcinomas and the age of onset is accelerated in homozygous versus hemizygous mice. However, the penetrance of the phenotype is incomplete, suggesting that the acquisition of additional mutations is necessary for tumorigenesis (15). In addition, the authors demonstrated that mice expressing a secretable form of EGF from the SP-C promoter develop alveolar hyperplasias also with incomplete penetrance. However, when crossed to the SP-C/c-myc animals, 100% of the double transgenic SP-C/c-myc/IgEGF animals develop bronchiolo-alveolar adenocarcinomas, suggesting cooperativity between the oncogenes, and an important role for EGF in promoting lung tumor growth. In order to examine the role of the retinoic acid receptor RARB2 in inhibiting lung tumorigenesis, Berard and colleagues (114) created a strain expressing an antisense RARB2 transgene under control of the MMTV promoter. A subset of the mice develop pulmonary adenomas and adenocarcinomas of the lung orginiating from type II cells or Clara cells as determined by immunohistochemistry.

Additional transgenic strains have elucidated genes whose mutation play a role in lung tumor formation, but are unable to induce tumorigenesis on their own. Mice heterozygous for deletion of the transforming growth factor b1 (TGF-beta 1) gene show an enhanced rate and increased multiplicity of lung tumorigenesis after treatment with carcinogen (44, 61). Furthermore, transgenic mice overexpressing a dominant-negative form of transforming growth factor beta receptor type II (TGF-beta RII) show an early increase in the incidence of lung tumors after treatment with carcinogen (3).

Diagnosis: Adenocarcinoma with neuroendocrine differentiation. Species: Mouse Boston reference set number: LW004

Until recently, there has been a great lack of mouse models that resemble human SCLC. Oncogenic H-ras driven from the neuroendocrine specific, calcitonin gene related protein (CGRP) promoter results in the development of both pulmonary neuroendocrine hyperplasia and non-NE tumors described as adenocarcinoma, with the later predominating (87). In an attempt to more closely mimic SCLC, Linnoila and colleagues expressed hASH1, a transcription factor involved in regulating NE differentiation, from the CC10 promoter (49). They found that in the context of coexpression of SV40 T Antigen from the CC10 promoter, CC10-hASH1 expression resulted in NE differentiation of airway epithelial cells, as well as the development of aggressive pulmonary NE carcinomas. Interestingly, CC10-hASH1 alone caused epithelial cell hyperplasia and metaplasia at the bronchioloalveolar junction, but did not result in NE differentiation of these cells. However, although these models develop neuroendocrine tumors, they may more closely resemble human NSCLC with neuroendocrine features than human SCLC.

Linnoila, Berns, and colleagues have now established a mouse model for neuroendocrine lung tumors by conditional inactivation of Rb1 and Trp53 in mouse lung epithelial cells. Mice carrying conditional alleles for both Rb1 and Trp53 developed high incidence aggressive lung tumors with morphologic and immunophenotypic similarities to human SCLC after intratracheal injection or intubation for delivery of Ad-Cre virus. The tumors, designated murine small cell lung carcinoma (MSCLC), stained positive for achaete-scute complex homolog-like 1 (Ascl1), Cgrp and synaptophysin. Pulmonary NE epithelial hyperplasia was evident 6-8 weeks after Cre administration, whereas the median latency of MSCLC was 210 days. MSCLCs had a marked capacity to metastasize to liver, brain, adrenal gland, bone and ovaries. This model will be a valuable tool for comparing human SCLC and MSCLC, identification of precursor lesions, and testing of targeted novel tumor intervention strategies and chemoprevention. (text modified from 123). Please see reference 123 for detailed information about this model and 124 for a mini-review.

In humans, lung cancer arises due to the accumulation of mutations in individual cells of the adult lung. The use of conditional alleles of oncogenes and tumor suppressor genes has facilitated the development of murine lung cancer models that more closely mimic the human situation, in which mutations occur in a subset of cells after completion of lung development. Cre/Lox technology has been used to develop conditional alleles of both oncogenes and tumor suppressor genes. Two strains of mice carrying conditional alleles of oncogenic K-ras G12D or K-ras G12V containing a floxed transcriptional stop element have been created. These mice develop pulmonary adenocarcinomas and epithelial hyperplasia of the bronchioles upon infection of the lungs with AdenoCre virus, a recombinant adenovirus expressing the Cre recombinase (38, 62). Lung tumor multiplicity can be regulated in these mice by altering the dose of virus administered. The use of AdenoCre virus to initiate tumorigenesis has facilitated the analysis of tumor progression as the precise timing of tumor initiation is known. In addition, the use of AdenoCre results in sporadic activation of the K-ras oncogene such that an individual tumor cell is surrounded by normal wild-type cells, more closely mimicking the development of human tumors. Another murine lung cancer model based on sporadic K-ras activation was developed using a variation of `hit-and-run' gene targeting (42). These mice carry a latent activatable allele of oncogenic K-ras G12D which is only expressed after a spontaneous somatic recombination event, resulting in the development of numerous pulmonary adenocarcinomas. The mice also develop intestinal aberrant crypt foci and skin papillomas perhaps reflecting the sensitivity of these tissues to the effects of K-ras mutations.

Several strains of mice have been created that carry floxed alleles of tumor suppressor genes some of which include condtional APC (83), NF1 (113), NF2 (27), Brca1 (5), Brca2 (43), p53 and RB (57). The use of AdenoCre virus or lung specific expression of Cre to inactivate one or a combination of relevant tumor supressor genes may be useful for the creation of additional conditional lung cancer models.

The tetracycline-based bitransgenic regulatable expression system has also been used for the creation of conditional murine lung cancer models. In this system, the reverse tet transactivator (rtTA), under the control of a tissue specific promoter, activates the expression of an oncogenic transgene under the control of the tet-operator. This system enables the investigator to both turn on and turn off oncogene expression at will, by administering or removing doxicycline (a tetracycline-related antibiotic). Both Sp-c-rtTa and Ccsp-rtTA transgenic mice have been created in order to direct expression of the tet-responsive gene to specific cells of the pulmonary epithelium. When Ccsp-rtTA mice are crossed to (tetO) 7-CMV-FGF-7 transgenic mice, postnatal administration of doxicycline results in the development of epithelial cell hyperplasia, adenomatous hyperplasia and pulmonary infiltration with mononuclear cells (94). Fisher and colleagues (21) created a (tetO)7-K-ras4b G12D mouse which they crossed to a Ccsp-rtTA trangenic strain. This Ccsp-rtTA strain suprisingly expressed rtTA primarily in alveolar type II cells, presumably due to transgene effects. The Ccsp-rtTA/(tetO)7-K-ras4b G12D bitransgenic mice develop multiple pulmonary adenocarcinomas only after administration of doxicycline. When bred into a background deficient for either the p53 or Ink4A/Arf tumor suppressor genes, the tumors arise more rapidly and appear more malignant. After withdrawal of doxicycline, the tumors rapidly regress, even in the absence of p53 or Ink4A, demonstrating that activated K-ras is necessary for both tumor initiation and maintenance (21).

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