Obesity Potentiates Esophageal Squamous Cell Carcinoma Growth and Invasion by AMPK-YAP Pathway

Abstract

Obesity could increase the risk of esophageal squamous cell carcinoma (ESCC) and affect its growth and progression, but the mechanical links are unclear. The objective of the study was to explore the impact of obesity on ESCC growth and progression utilizing in vivo trials and cell experiments in vitro. Diet-induced obese and lean nude mice were inoculated with TE-1 cells, then studied for 4 weeks. Serum glucose, insulin, leptin, and visfatin levels were assayed. Sera of nude mice were obtained and then utilized to culture TE-1. MTT, migration and invasion assays, RT-PCR, and Western blotting were used to analyze endocrine effect of obesity on cell proliferation, migration, invasion, and related genes expression of TE-1. Obese nude mice bore larger tumor xenografts than lean animals, and were hyperglycemic and hyperinsulinemic with an elevated level of leptin and visfatin in sera, and also were accompanied by a fatty liver. As for the subcutaneous tumor xenograft model, tumors were more aggressive in obese nude mice than lean animals. Tumor weight correlated positively with mouse body weight, liver weight of mice, serum glucose, HOMA-IR, leptin, and visfatin. Obesity prompted significant TE-1 cell proliferation, migration, and invasion by endocrine mechanisms and impacted target genes. The expression of AMPK and p-AMPK protein decreased significantly (P < 0.05); MMP9, total YAP, p-YAP, and nonphosphorylated YAP protein increased significantly (P < 0.05) in the cells cultured with conditioned media and xenograft tumor from the obese group; the mRNA expression of AMPK decreased significantly (P < 0.05); YAP and MMP9 mRNA expression increased significantly (P < 0.05) in the cells exposed to conditioned media from the obese group. In conclusion, the altered adipokine milieu and metabolites in the context of obesity may promote ESCC growth in vivo; affect proliferation, migration, and invasion of ESCC cells in vitro; and regulate MMP9 and AMPK-YAP signaling pathway through complex effects including the endocrine effect.

Figure 1

MMP9 protein expression in ESCC cell lines. ^∗P < 0.05 versus TE-1.

Figure 2

Subcutaneous tumor xenograft growth curve. Tumor xenografts from obese nude mice grew faster than those from lean and nonobese animals within 4 weeks. ^∗P < 0.05 versus lean and nonobese, ^∗∗P > 0.05 versus nonobese.

Figure 3

Liver character from obese and lean mice. (a) Unaided observation of the liver showed that the color of the liver became more yellow. (b) Oil red O staining of the liver specimen under a microscope demonstrated that lipid in cytoplasm became red with the help of oil red O, and the fields of the liver specimen from the obese group get more red than that from lean mice. Magnification, ×400.

Figure 4

MMP9, AMPK, and YAP protein expression in these subcutaneous tumors was evaluated semiqualitatively. ^∗P < 0.05 versus lean.

Figure 5

TE-1 cell proliferation in different states. TE-1 cultured with conditioned medium from sera of obese mice grew faster than those from lean or RPMI-1640. ^∗P < 0.05 vs. lean.

Figure 6

Effects of obesity on TE-1 cell migration. Obesity prompted significantly TE-1 cell migration than the lean (270.1 ± 19.8 versus 158.0 ± 21.6/field, ^∗P < 0.001). Magnification, ×200.

Figure 7

Effects of obesity on TE-1 cell invasion. Obesity accelerated significantly TE-1 cell invasion than the lean (234.0 ± 26.9 versus 112.4 ± 18.9/field, ^∗P < 0.001). Magnification, ×200.

Figure 8

Related mechanism that diet-induced obesity accelerated TE-1 cell growth and progression was dissected. Obesity prompted the growth and development of TE-1 cells by MMP9 and AMPK-YAP signaling pathway at the mRNA level by RT-PCR (a, b) and at the protein level by Western blotting (c, d). ^∗P < 0.05 versus lean.