Esophageal cancer is the eighth most common cancer worldwide (with 482,000 new cases) and the sixth most common cause of death from cancer (with 407,000 deaths estimated in 2008) (1
). Esophageal squamous cell carcinoma (ESCC) is the predominant subtype. Because of an absence of early symptoms, ESCC is frequently diagnosed at an advanced stage of the disease. Although cancer mortality rates have declined worldwide since the mid 1980s, ESCC remains a deadly cancer with a 5-year survival of only 10%. Thus, insights into its pathogenesis are critical in order to devise new preventions, earlier diagnostics, and novel treatment strategies.
Epidemiological and clinical studies show that chronic inflammation predisposes to many types of cancer, including ESCC (2
). In this regard, persistent inflammation in the esophagus is a frequent occurrence in populations at high risk for ESCC (5
). The challenge is to determine the cause of this chronic inflammation and its role in ESCC development.
The major risk factors for ESCC are chronic alcohol consumption, tobacco use, nutritional deficiencies, and exposure to environmental carcinogens such as N
-nitrosomethylbenzylamine (NMBA) (6
). In particular, zinc (Zn)-deficiency (ZD) is implicated in the pathogenesis of ESCC in many populations (8
), including persons with chronic alcohol consumption (10
). Abnet et al. (8
) provided the strongest evidence of an association between dietary ZD and ESCC in a high incidence area by establishing an inverse relationship between Zn concentration in biopsy samples and the subsequent risk of developing ESCC. Although Zn is present in a large variety of foods, its content is low in most, with the exception of red meat and seafood. Accordingly, individuals subsisting largely on a cereal diet are likely to be Zn-deficient. In the US 10% of the population is estimated to ingest less than 50% of the recommended daily allowance for Zn (11
). In the developing world, dietary ZD may affect more than 2 billion people (12
). Because Zn is required for the activity of many enzymes, for proper immune function, and for the conformation of many transcription factors that control cell proliferation, apoptosis, and signaling pathways (13
), ZD predisposes to disease by adversely affecting these processes.
The environmental carcinogen NMBA is widely used to induce esophageal tumors in rodents. An early study reported that NMBA induces a 63% incidence of ESCC in nutritionally complete rats after a cumulative dose of 50 mg/kg body weight (2.5 mg/kg weekly for 20 weeks) (16
). A typical esophageal tumor bioassay in chemoprevention studies entails weekly administration of low doses of NMBA for 15 weeks (cumulative dose = 7.5 mg/kg), producing a 100% incidence of squamous papillomas but without ESCC development (17
). The tumorigenicity of NMBA in rodents is the results of the bioactivation of the carcinogen by esophageal cytochrome P450 isozymes to produce a DNA-methylating agent, leading to the formation of the mutagenic DNA adduct O6
), and a high prevalence of mutations in Ha-Ras and p53 genes in papillomas (19
Our ZD rat esophageal tumor model that combines dietary ZD with exposure to the environmental carcinogen NMBA (20
) mimics aspects of human ESCC in high-risk populations (6
). We have shown in prior studies that weanling rats on a ZD diet for ~6 weeks develop increased cell proliferation and changes in gene expression in the squamous epithelium, including overexpression of Cox-2
). The hyperplastic ZD rat esophagus is highly sensitive to NMBA (20
), displaying a 93% incidence of esophageal papillomas (but no ESCC) after a single exposure to a low NMBA dose (2 mg/kg). Whether dietary ZD could promote ESCC development in tumor bioassays by low NMBA doses has not been determined.
DNA microarray analysis using genome arrays provides a powerful tool to understand how ZD might affect gene expression to predispose to esophageal carcinogenesis. Using a rat genome array, we reported that short-term ZD (6 weeks) alone (without NMBA) induces a distinct gene signature in the esophageal mucosa with upregulation of two proinflammation-genes S100 calcium binding protein a8 and a9
, associated with esophageal epithelial hyperplasia. Zn was shown to modulate the interaction of S100A8 with its receptor for advanced glycation end products (RAGE) and the downstream nuclear factor (NF)-kB-COX-2 signaling pathway (22
We now ask whether prolonged ZD might amplify the inflammatory program to provide a microenvironment conducive to ESCC development on exposure to low carcinogen doses, thus serving as a faithful model for conditions that lead to human ESCC. For this, we conducted a long-term tumor bioassay in Zn-modulated rats using low doses of NMBA. In parallel, we performed transcriptome profiling of esophageal mucosa at tumor endpoint and during tumor development in order to correlate gene expression changes with tumor progression.