Increased B7-H4 expression during esophageal squamous cell carcinogenesis is associated with IL-6/STAT3 signaling pathway activation in mice

Abstract

B7-homolog 4 (B7-H4), one of the costimulatory molecules of the B7 family, has been reported to be widely expressed in multiple types of cancer tissues, and to be important in tumor progression and poor prognosis. However, the role of B7-H4 in esophageal precancerous lesions has not been elucidated yet. In the present study, a model of esophageal squamous cell carcinogenesis was established in 208 C57BL/6 mice by 4-nitroquinoline-1-oxide (4NQO) drinking water of mice, and the changes in the expression of B7-H4 during the whole pathological process were investigated. Hematoxylin and eosin staining results demonstrated that the pathological stage was exacerbated with the increase in time of 4NQO-mediated carcinogenesis induction, and the pathological features were similar to those observed in humans. Immunohistochemistry results revealed that B7-H4 expression was upregulated and positively correlated with pathological stage (P<0.0001) as well as with infiltration of cluster of differentiation (CD) 11b⁺ macrophage cells (P=0.0002). In addition, B7-H4 messenger RNA expression increased in the esophagi of model mice compared with that of control mice, which was positively associated with the gene expression of interleukin (IL)-6 and signal transducer and activator of transcription 3 (STAT3), according to the results of reverse transcription-quantitative polymerase chain reaction analysis. Similarly, B7-H4 protein expression was upregulated in the esophageal tissues of model mice in comparison with that of control mice, and was positively associated with IL-6 expression and STAT3 phosphorylation. In conclusion, the present data suggested that B7-H4 expression increased during esophageal squamous cell carcinogenesis in mice in association with IL-6/STAT3 signaling pathway activation.

Keywords: 4-nitroquinoline-1-oxide; B7-homolog 4; esophageal carcinogenesis; interleukin-6/signal transducer and activator of transcription 3 signaling pathway; precancerous condition.

Figure 1.

Changes in body weight and esophageal pathological conditions during squamous cell carcinoma formation induced by 4NQO in C57BL/6 mice. (A) The body weight of female and male 4NQO model mice increased more slowly than that of control mice (*P<0.05, **P<0.01, Student's t-test). (B) The esophageal pathological conditions of 4NQO model mice were exacerbated during the carcinogenesis process. (C) Hematoxylin and eosin staining of esophageal precancerous lesions (magnification, ×400). LGIN, low-grade intraepithelial neoplasia; HGIN, high-grade intraepithelial neoplasia; 4NQO, 4-nitroquinoline-1-oxide.

Figure 2.

B7-H4 expression, and CD4⁺T, CD8⁺T and CD11b⁺ macrophage cell infiltration in esophagi of mice, as estimated by immunohistochemistry staining. B7-H4 was expressed in the membrane and cytoplasm of epithelial cells in the mucosa layer. Infiltration of CD4⁺T, CD8⁺T and CD11b⁺ macrophage cells was predominantly located in the submucosa of esophageal tissues. Magnification, ×400. The arrows indicate the cells that are positive. B7-H4, B7-homolog 4; CD, cluster of differentiation.

Figure 3.

Gene expression of B7-H4 and associated molecules in control mice. The expression of genes in the esophagi of control mice did not change obviously from weeks 16 to 28 (B7-H4: P=0.0650; IL-6: P=0.2842; IL-10: P=0.2816; TGF-β, P=0.0743; IFN-γ: P=0.3399; STAT3: P=0.3225; Kruskal-Wallis analysis). mRNA, messenger RNA; Cq, quantification cycle; B7-H4, B7-homolog 4; CD, cluster of differentiation; IL, interleukin; TGF, transforming growth factor; IFN, interferon; STAT3, signal transducer and activator of transcription 3.

Figure 4.

Gene expression of B7-H4, IL-6, IL-10, TGF-β and STAT3 in the esophagi of 4NQO model mice increased during the carcinogenesis process (*P<0.05, **P<0.01, vs. the control group; Kruskal-Wallis analysis). mRNA, messenger RNA; Cq, quantification cycle; B7-H4, B7-homolog 4; CD, cluster of differentiation; IL, interleukin; TGF, transforming growth factor; IFN, interferon; STAT3, signal transducer and activator of transcription 3; 4NQO, 4-nitroquinoline-1-oxide.

Figure 5.

Expression of B7-H4 and associated proteins in control mice. Protein expression in the esophagi of control mice did not change obviously from weeks 16 to 28 (all P>0.05, Kruskal-Wallis analysis). B7-H4, B7-homolog 4; IL, interleukin; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated.

Figure 6.

B7-H4, p-STAT3 and IL-6 protein expression in the esophagi of 4NQO model mice increased during the carcinogenesis process (*P<0.05, **P<0.01 vs. the control group; Kruskal-Wallis analysis). B7-H4, B7-homolog 4; IL, interleukin; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated; 4NQO, 4-nitroquinoline-1-oxide.