Animal models of head and neck cancer, especially oral cancer, have been used extensively to study chemical carcinogens as elaborated upon in the previous section. However, genetic approaches have lagged considerably until very recently through the employment of gene overexpression in transgenic mice and targeted gene ablation in embryonic stem cells of mice. The advantages of genetic models in mice are several, including the ability to recapitulate human carcinogenesis in large numbers of animals, dissect the molecular basis for the different stages of carcinogenesis, availability of mouse-specific molecular reagents, and application of novel chemopreventive and therapeutic approaches.
A. Targeting Genes to Oral Cavity Epithelia with Promoters
Because the oral cavity is lined by a stratified squamous epithelium, there are certain immediate considerations. This is the incorporation of promoters that have been used predominantly for skin models. These include viral promoters, such as human papillomavirus (HPV) E6 and E7, and cellular promoters, i.e., genes that are associated with basal and suprabasal cells. In the latter context, promoters for keratins 5 and 14 will lead to the targeting of genes to basal cells. Seven hundred base pairs of the 3' downstream sequence was used to drive the expression of an intronless human K14 gene in transgenic mice, and the construct was expressed in a fashion analogous to the endogenous K14 gene: in the basal layer of stratified squamous epithelia , Suprabasal promoters include those for keratins 4 and 13 (preferred partners in oral cavity, whereas in skin it is keratins 1 and 10) as well as for involucrin . Six thousand base pairs of the 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia . However, only 90 bp of the K5 promoter directs expression to stratified epithelia, with expression predominantly in the epidermis, hair follicles, and tongue.
Other promoters studied include adenosine deaminase (ADA), which is highly expressed in four tissues of the mouse: the maternal decidua, the fetal placenta, the keratinizing epithelium of the upper alimentary tract (tongue, esophagus, and forestomach), and the absorptive epithelium of the proximal small intestine [67,68]. However, ADA is produced at relatively low levels in all other tissues.
A viral promoter may prove to be of broad appeal in recapitulating oral carcinogenesis. The Epstein-Barr virus (EBV) contains nearly 170 kb of the genomic sequence. Most genes are involved in viral replication; however, some play a role in the ability of the virus to immortalize B
lymphocytes. Nearly 800 bp of the ED-L2 promoter, located 3' to the LMP-1 gene, regulates heterologous reporter genes in cell lines derived from the esophagus and oral cavity, but not of other cell lineages, including B lymphocytes (Fig. 5.1) . In turn, the promoter is regulated transcriptionally in a complex interplay of tissue-specific and relatively tissue-restricted transcriptional factors [70-72]. Given these findings, the EBV ED-L2 promoter has been linked to the human cyclin D1 cDNA and utilized this transgene to generate founder lines. This transgene is transcribed specifically in the tongue, esophagus, and forestomach, all sharing a stratified squamous epithelium. The transgene protein product localizes to the basal and suprabasal compartments of these squamous epithelial tissues, and mice from different lines develop dysplasia, a precursor to carcinoma: mild dysplasia by 6-8 months of age and moderate-severe dysplasia by 16-18 months of age in contrast to age-matched wild-type littermates. Furthermore, the dysplastic phenotype is associated with increased cell proliferation based on PCNA overexpression and abnormalities in cyclin-dependent kinase 4 (cdk4), epidermal growth factor receptor (EGFR), and p53 , In aggregate, these studies suggest that alterations in certain oncogenes and tumor suppressor genes occur early during oral carcinogenesis.
More recently, we have bred the ED-L2 cyclin D1 mice with p53-deficient mice. Importantly, the cyclin Dl/p53 heterozygous mice develop severe dysplasia by 3-6 months in the oral-esophageal epithelium and histologic evidence of invasive cancer by 12 months (unpublished observations by A. Rustgi and colleagues). The cyclin Dl/p53 null mice also develop severe dysplasia by 3-6 months in the oral-esophageal epithelium with suggestions of microinvasion; however, it proves to be difficult to observe these mice beyond about 6 months due to the fact that they succumb to systemic sarcomas or lymphomas, the expected phenotype of the p53 null alone genotype.
B. Other Emerging Genetic Models of Head and Neck Cancer
In addition to the ED-L2 transgenic mouse model, a number of mouse-based genetic models for head and neck cancer are currently being developed. These efforts are primarily ongoing in the molecular carcinogenesis units of the Oral and Pharyngeal Cancer Branch (OPCB) at the National Institute of Dental and Craniofacial Research under the leadership of J. Silvio Gutkind.
Adopting an approach that has been developed in Harold Varmus' laboratory while at NIH [74-76], which first targets the avian leucosis virus receptor, tv-a, to the target tissue of interest and then uses highly effective avian retroviruses to carry the transgene and target its expression in vivo in a tissue-specific manner. Investigators at the OPCB have created a transgenic mouse line carrying the tv-a receptor gene to the basal layer of stratified epithelium using the cytokeratin 5 (K5) promoter with the hope of being able to eventually induce tumor development on sequential or coexpression of known oncogenes and dominant-negative tumor suppressors. The advantages of this mouse genetic model include (1) the targeting tissue-specific transgene expression without the need to generate a new mouse line; (2) the ability
EBV ED-L2 promoter
Cyclin D1 cDNA
EBV ED-L2 promoter
Cyclin D1 cDNA
to sequentially target multiple transgene to the same host; (3) the ability to monitor the fate of individual, genetically modified cells; (4) the ability to combine oncogenic and tumor suppressor alterations in a specific tissue/cell type to provide insights regarding the effects of different mutations in carcinogenesis and recapitulating the multistep nature of tumorigenesis; and (5) the ability to test the role of newly discovered candidate molecules (e.g., by gene expression profiling analysis) in head and neck cancer carcinogenesis.
The OPCB has also generated a K5 transgenic mouse capable of inducing the expression of transgenes to the basal layer of stratified epithelium. This mouse line carries the tetracycline-inducible promoter system (tet-on receptor) with the eventual hope of being able to induce the expression of candidate oncogenes under the control of the tetracycline responsive promoter.
A third mouse model currently developed by this group utilizes the sprr3 promoter. This is a member of the small proline-rich family of proteins that is expressed preferentially in the oral epithelium. The sprr3 promoter is being explored to target transgenes (oncogenes and dominant tumor suppressors) to oral epithelium.
Given the relative tissue-specific expression of keratin 4, targeted disruption of this gene through homologous recombination in mouse embryonic stem cells has a striking phe-notype of epithelial hyperplasia in the tongue and esophagus, suggesting an impairment in differentiation . This is evident in K4 null mice as early as 2 months, but K4 heterozygous mice and wild-type mice are normal. No cancer develops, perhaps due to compensation of K4 loss by K13. Interestingly, mice lacking involucrin from embryonic stem cells develop normal tissue structures.
The ED-L2 promoter will be a powerful tool to target genes to the oral cavity, either singly or in combination in transgenic mice. Furthermore, this promoter may be useful in strategies for conditional knockout of oncogenes and tumor suppressor genes. Apart from these considerations, oral cavity-specific genes may emerge from ongoing efforts in microarray approaches with subsequent application in genetically engineered animal models.
Mouse genetic models will have a particularly important role in head and neck cancer research. These models will permit the site-specific interactions of oncogenes and tumor suppressors in the head and neck region. Another utility of these models will be to validate and test the role of newly discovered molecules in head and neck cancer carcinogenesis. This is the basis of the integrative human-based discovery and animal model testing for head and neck cancer research.
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