Comparable molecular alterations in 4-nitroquinoline 1-oxide-induced oral and esophageal cancer in mice and in human esophageal cancer, associated with poor prognosis of patients.

BACKGROUND: The murine model of 4-nitroquinoline 1-oxide (4-NQO)-induced oral and esophageal cancer is frequently used to assess the effects of different cancer prevention/ therapy agents in vivo, but the molecular mechanisms in those 4-NQO-induced carcinogenesis are unknown. This study investigated aberrant expression of cell growth-critical genes in 4-NQO-induced oral and esophageal cancer tissues in mice compared to those present in the human disease for association with survival of patients.
MATERIALS AND METHODS: C57LB6/129Sv mice were given 4-NQO in their drinking water to induce oral and esophageal cancer. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry were used to detect gene expression in the cancer tissues from mice and in 4-NQO-treated human esophageal cancer cell lines and esophageal cancer tissues. Methylation-specific PCR and DNA sequencing were performed to assess methylation of the Rarb2 promoter in murine tissues. Kaplan-Meier analysis was performed to associate gene expression in esophageal cancer tissues with survival data for patients with esophageal cancer.
RESULTS: 4-NQO dose-dependently induced pre-malignant and malignant lesions in the oral cavity and esophagus in mice that pathologically and morphologically mimicked human oral and esophageal cancer. Molecularly, 4-NQO inhibited Rarβ₂ but induced expression of phosphorylated extracellular-signal-regulated kinase-1 and -2 (p-ERK1/2) and Cox2 proteins and Rarβ₂ gene promoter methylation in murine tumors. In vitro treatment with 4-NQO altered expression of RARβ₂, p-ERK1/2, and COX2 in human esophageal cancer cells. In tissues from 90 patients with esophageal cancer, expression of p-ERK1/2 and COX2 was up-regulated, and p-ERK1/2 expression was associated with advanced clinical tumor stage and consumption of hot beverages, while COX2 expression was associated with tumor de-differentiation in esophageal cancer. Furthermore, expression of p-ERK1/2 was associated with a worse overall survival rate of patients (p=0.014), whereas the association of COX2 expression with worse overall survival rate did not reach statistical significance (p=0.19). Knockdown of COX2 expression using transient transfection of a COX2 antisense expression vector inhibited Ki67 expression, an indicator of cell proliferation, in human esophageal cancer cells.
CONCLUSION: 4-NQO-induced cancer in oral cavity and esophagus of mice not only pathologically and morphologically mimicked human oral and esophageal cancer, but also shared some molecular alterations (e.g. aberrant expression of Rarb2, p-ERK1/2, and Cox2). This study further demonstrated that targeting of the altered RARβ₂-led gene pathway could effectively suppress the development of this deadly type of cancer.