Oral-Specific Chemical Carcinogenesis in Mice: An Exciting Model for Cancer Prevention and Therapy Free

Editor's Note: Preclinical animal models that are clinically relevant to and suitable for cancer chemoprevention research are beginning to emerge. Because of the critical importance of these models, we are publishing this second perspective on the modeling article by Czerninski et al. in this issue of the journal. Whereas the perspective by Dennis focuses on translating mammalian target of rapamycin targeting in animal models into clinical preventive drug development, this “different” perspective focuses on strengths of the new mouse model of Czerninski et al.—oral specificity and chemical induction and diversity of heterogeneity of lesions—as a clinically relevant animal model that overcomes certain limitations of other animal models for oral cancer chemoprevention.

Humans are constantly bombarded with physical, chemical, and biological insults—such as UV radiation, toxic chemicals, and pathogens—that can damage DNA, activate oncogenic pathways, and cause inflammation. These damages can lead to precancerous lesions that can become malignant. Whether such precancerous lesions can be prevented and whether the progression of precancerous lesions to malignant neoplasms can be controlled have been the subjects of intense research. Despite great interest in cancer chemoprevention, however, relatively few animal models have been developed or are available for testing chemopreventive strategies. In this issue of the journal, Czerninski et al. (1) describe an oral-specific chemical carcinogenesis mouse model that shows the development of precancerous lesions into malignant squamous cell carcinomas (SCC). The relevance of this oral-specific model to cancer prevention research is substantiated by its pathologic features and molecular alterations including the activation of epidermal growth factor receptor (EGFR), cyclooxygenase-2, and the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in tumors, which recapitulate what has been found in clinical samples. The model could provide a new platform for validating molecular-targeted chemopreventive strategies (1).

Czerninski et al. (1) treated mice with 4-nitroquinoline-1 oxide (4NQO), a DNA adduct-forming agent causing DNA damage mimicking that induced by cigarette smoke, for 16 weeks. At the end of 4NQO treatment, 100% of the mice exhibited precancerous lesions and/or tumors in their tongues and oral mucosa. Many of these lesions progressed to malignant SCC even after 4NQO was withdrawn. When the mice were treated with rapamycin, however, the progression of oral precancerous lesions to malignant SCC was greatly reduced; more striking, some SCCs regressed. Therefore, Czerninski et al.'s study supports further developing drugs that target the mTOR pathway to prevent precancerous lesions from progressing to malignant SCC in the oral cavity, and potentially, other tumor types with deregulated mTOR pathway. Testing whether precancerous lesions can be prevented if mice are treated at an earlier stage is one attractive possibility; for instance, mice could be treated with rapamycin before 4NQO treatment or at various time points during 4NQO treatment and before visible lesion development. Because Fenton and Hord (2) have shown that chemopreventive strategies can depend on the stage of the cancer, such studies could further ascertain whether rapamycin can be used to reduce or even inhibit carcinogen-induced precancerous lesions and tumors at different stages.

Several xenograft or genetically defined mouse models, such as the phosphatase and tensin homologue (PTEN) heterozygous mouse model for endometrial hyperplasia (3) and Apc heterozygous mouse model for intestinal neoplasia (4), have been exploited for chemopreventive research. Although these models are valuable for studying chemoprevention in high-risk patients, they lack the important clinical feature of tumor heterogeneity. Therefore, chemical carcinogenesis models better reflect the clinical setting because of the diversity and heterogeneous nature of the resulting tumoral lesions. Other chemical carcinogenesis models have been studied for chemoprevention, including urinary bladder cancer (5) and lung cancer (6) models. The oral-specific chemical carcinogenesis model of Czerninski et al., however, is more attractive for chemoprevention study because of its easier utility for observation and tissue sampling for pathologic and molecular analyses. Furthermore, oral epithelium could be used as a surrogate tissue for assessing smoking-induced molecular alterations in the lungs (7).

Approximately 500,000 patients are diagnosed with head and neck SCC (HNSCC) each year, making this the sixth most common cancer worldwide. Advances in molecular biology and genomic technologies have uncovered many deregulated pathways and mutations in HNSCC (Fig. 1), in which the most frequently detected gene mutations are listed in Table 1. Five or six genetic or epigenetic changes must occur before a normal human cell transforms into an invasive and subsequently metastatic cancer cell (7). One frequently found alteration is activation of the phosphatidylinositol-3-OH kinase/PTEN/mTOR pathway (Fig. 1), which controls cell metabolism, size, proliferation, and apoptosis. Drugs that target AKT, phosphatidylinositol-3-OH kinase, and mTOR are being developed for cancer prevention and treatment (8–10). The AKT-mTOR pathway was activated in the Czerninski et al. mouse model. Moreover, increased cyclooxygenase-2 expression was detected in early dysplastic lesions and was even more apparent in SCCs. In addition, EGFR overexpression was observed in multifocal areas throughout the tumor. These molecular events mimic those of human HNSCC (9, 11, 12).

Numerous studies have focused on identifying the genetic and epigenetic alterations underlying HNSCC (13–16). Further mutational and genomic analysis of precancerous lesions and tumors induced in the oral-specific mouse model could unveil which lesions are most susceptible to specific chemopreventive agents; for example, whether oral lesions with p53 mutations or cyclin-dependent kinase 2A mutations have enhanced susceptibility to rapamycin. Addressing such questions in the oral-specific model may translate into markers that can be used to stratify patients in clinical trials.

After its discovery more than a decade ago (17), mTOR has been established as a vital controller of overall cell growth through integrating signals from hormones, growth factors, nutrients, energy, and other environmental factors. Numerous elements of the mTOR signaling pathway are dysregulated in precancerous lesions, and inhibition of the mTOR pathway is very appealing in preventive settings. Czerninski et al.'s findings further support targeting the mTOR pathway as a chemoprevention strategy. Other potential targets for chemoprevention besides mTOR (18) include AKT (14, 19, 20), cyclooxygenase-2, and EGFR (21, 22). The overexpression and activation of these molecules in precancerous lesions and tumors in this mouse model suggests its usefulness for evaluating new drugs that modulate these targets. Furthermore, the expression of the upstream regulators and downstream targets of AKT, mTOR, and cyclooxygenase-2 are altered in multiple human tumors. For example, increased AKT activity is a result of human EGFR-2 (HER2/neu) overexpression (23). Phosphatidylinositol-3-OH kinase amplification and overactivation have been detected in ∼40% of head and neck tumors (16). Similarly, inactivation of tuberous sclerosis protein 2 has been found to activate the expression of mTOR signaling in renal tumors (24). Following up on these tumor analyses, a more in-depth genetic and genomic characterization of precancerous lesions in this oral-specific mouse model may fully realize its potential.

We are at a crossroads between chemoprevention and chemotherapy. With advances in molecular and genomic technologies, many dysregulated pathways have been identified in malignant and precancerous tissues; chemotherapy and chemoprevention are targeting the opposite ends of a continuum. Specific molecular-targeted agents for chemotherapy are becoming increasingly available, and we can now test these new agents for chemoprevention. Many chemotherapeutic agents targeting specific signaling molecules might be applicable to chemoprevention because many signaling molecules are dysregulated in both cancers and precancerous lesions. However, these chemotherapeutic drugs are usually administrated at toxic doses not suitable for chemoprevention, where agents generally are administrated for longer periods of time in relatively healthy people and so must be highly tolerable. The route of application is equally important to chemoprevention. Despite Czerninski et al.'s successful proof-of-principle studies of rapamycin, injecting rapamycin likely will not be acceptable in clinical prevention trials. Perhaps the well-developed oral mTOR inhibitor, everolimus, would be a more promising alternative. As supported by proof-of-principle studies of budesonide in laboratory animals with lung cancer (25, 26) and in patients with bronchial dysplasia (27), inhalational exposure to chemopreventive agents is also a promising strategy for chemoprevention.

Several dietary compounds such as genistein, vitamin E succinate, curcumin, and resveratrol have potential for cancer chemoprevention because of anticarcinogenic activity (28) and relatively low acute toxicity. Liposomal curcumin suppresses HNSCC growth in vitro and in vivo via an AKT-independent pathway (29). Vitamin E succinate also inhibits HNSCC growth and viability in vivo and in vitro (30). Testing these agents alone and in combinations (possibly including mTOR inhibitors) in the oral-specific mouse model would be a logical step in evaluating their potential for oral cancer prevention.

The development of oral cancer prevention agents has been limited heretofore by a lack of clinically relevant preclinical oral carcinogenesis models. The best-studied in vivo oral model is the decades-old model of 7,12-dimethylbenz[a]anthracene-induced carcinogenesis in the hamster cheek pouch. This model induces ras mutations, however, which have little relevance to clinical oral carcinogenesis in the U.S. and many other countries. There are recently developed gene-based animal models, the best of which involves ras mutations and deletion of transforming growth factor-beta II receptor (31, 32). This model also is limited by ras mutations as well as by a lack of lesion and tumor heterogeneity. 4NQO models generally develop esophageal cancer and its mortality prior to an oral cancer endpoint. Czerninski et al. have optimized the 4NQO model by studying different administration routes and doses of 4NQO in different mouse strains, thus overcoming the interference of esophageal cancer and allowing an oral cancer endpoint. This model is simple, fast and reproducible and has molecular heterogeneity (e.g., altered EGFR, COX-2 and p53) that is consistent with human oral carcinogenesis.

In conclusion, Czerninski et al. have developed a relevant model for preclinical chemoprevention studies. Tumor heterogeneity in this model will help in understanding the complexity of tumor initiation and progression in humans. However, a detailed molecular characterization of the heterogeneity of premalignant and malignant tissues in the model will be necessary to fully realize its potential for chemoprevention. A further advance would be the introduction of specific genetic alterations within the oral-specific chemical carcinogenesis model in order to better study mechanisms of molecular targeted drugs, drug resistance, and biology with potential relevance to high-risk human subgroups.