Genetic alteration of stemness factors and p53 in mouse forestomach by chemical carcinogen-induced carcinogenesis

Abstract

The expression profiles of genes normally enriched in embryonic stem (ES) cells (stemness factors) are associated with poor clinical outcome in solid tumors. However, whether such gene expression is responsible for tumor initiation and progression remains to be determined. The tumor suppressor gene p53 is known to attenuate the expression of Nanog, which is essential for maintaining stem cells in response to DNA damage. On the basis of these findings, we hypothesized that stemness factors and p53 closely correlate with each other and form a network in response to genomic damage in the early phase of carcinogenesis and in the process of tumor progression. In this study, we applied the N-methylbenzylnitrosoamine (NMBA)-induced carcinogenesis model to the mouse forestomach to clarify the role of the stemness factors, c-Myc, Klf4, Sox2, Oct3/4 and Nanog, in cancer development using p53(+/+) (n=26) and p53(+/-) (n=11) C57BL/6J mice. Thirty weeks following NMBA administration, histologically evident squamous cell carcinoma was detected in the forestomachs of p53(+/+) mice, and the percentage of p53-positive nuclei in the forestomach epithelium gradually increased during carcinogenesis. Tumor development in p53(+/-) mice occurred significantly earlier than in p53(+/+) mice. Quantitative real-time PCR analyses revealed a reduced c-Myc and Klf4 expression before evident morphological changes were observed, and an increased expression with the development of squamous cell carcinoma. Sox2 expression remained unchanged until tumor development and increased with the appearance of squamous cell carcinomas. The expression of Oct3/4 and Nanog increased at the early stages following NMBA administration, and Nanog expression in situ was not positively affected by the deficiency of p53. Findings of the present study suggested that Oct3/4 may be involved in the progression of carcinogenesis from normal epithelial cells at early stages, suggesting the potential use of Oct3/4 as a biomarker in forestomach tumor formation at early stages of chemical carcinogenesis.

Figure 1

Macroscopic and histological appearance in C57BL/6J p53+/+ forestomach following NMBA administration. A: Macroscopic appearance. A total of (a) 4 weeks, (b) 8 weeks, (c) 12 weeks, (d) 20 weeks and (e) 30 weeks following NMBA treatment. Thickened forestomach and elevated tumors were detected from 20 weeks following NMBA administration. B: Histological appearance of H&E-stained sections. (a) Normal forestomach epithelium without NMBA treatment. (b and d) Twenty weeks following NMBA administration, the sections demonstrated intraepithelial hyperplasia. (c and e) Thirty weeks following NMBA administration, there was an evident squamous cell carcinoma histologically in the forestomach that had invaded into the submucosal layer.

Figure 2

Immunohistochemical staining of p53 in C57BL/6J p53+/+ forestomach following NMBA administration. A: (a) P53 staining of normal mice forestomach without NMBA treatment. A total of (b) 4 weeks, (c) 12 weeks and (d) 30 weeks following NMBA administration is shown. B: The percentage of p53-positive nucleus in the forestomach epithelium. The p53 expression was gradually increased, and in 30 weeks following NMBA administration in mice, p53 was overexpressed in squamous cell carcinoma lesions; n=5 for each group.

Figure 3

Quantitative real-time PCR analysis of stemness factors (c-Myc, Klf4, Sox2 and Oct3/4) relative to GAPDH were performed by using the bulk RNA extracted from the C57BL/6J p53+/+ forestomach. PCR analysis of Nanog was specifically conducted by using the C57BL/6J p53+/+ and C57BL/6J p53+/- forestomach; n=5 for each group.