Purpose:

We previously revealed that the expression of Annexin A2 (ANXA2) was increased in ESCC by two-dimensional electrophoresis and MALDI-TOF and LC-ESI-IT MS. This study aims to investigated the implication of Annexin A2 in ESCC.

Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription.

Experimental Design:

Immunohistochemistry was performed to analyze the expression of ANXA2 in esophageal squamous cell carcinomas (ESCC). siRNA knockdown, transwell assay and wound-healing assays were used to investigate the function of ANXA2 in cells. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis were used to seek the potential effector molecule of ANXA2. Chromatin immunoprecipitation, and reporter gene assays were used to confirm the relationship between c-Myc and HIF-1 alpha. Co-immunoprecipitation assay was performed to demonstrate the relationship between ANXA2 and c-Myc. Immunofluorescence was used to reveal the location of ANXA2.

Results:

Knockdown of Annexin A2 significantly inhibited the migration and invasion of ESCC cells. At the molecular level, VEGF, MMP-2, MMP-9, p-MEK and p-ERK were down-regulated in ANXA2-siRNA cells. The HIF-1 alpha mRNA and protein levels were significantly lowered after knocked down the Annexin A2. HIF-1 alpha is the effector molecule of ANXA2 in ESCC cells, and that ANXA2 affects the expression of HIF-1 alpha at the transcription level because when knocking down HIF-1 alpha, the level of VEGF, MMP-2, MMP-9, p-MEK and p-ERK reduced as well. c-Myc is a transcription factor for HIF-1 alpha. ANXA2 has a direct protein interaction with c-Myc and knockdown of ANXA2 down-regulated the expression of c-Myc. The Tyr 24 phosphorylation form of Annexin A2 (Y24D-ANXA2) was located in the nucleus, and the non-phosphorylation form (Y24A-ANXA2), in the cytoplasm and cell membrane. Transfection of pcDNA3.1-ANXA2-Y24D enhanced the migration and invasion as compared with that of pcDNA3.1-ANXA2-Y24A in esophageal cancer cells. A treatment with the Annexin A2 phosphorylation inhibitor, Dasatinib, changed the location of Annexin A2 and lowered the expression of c-myc and HIF-1 alpha.

Conclusions:

These results suggest that in esophageal cancer cells, phosphorylated Annexin A2 could enter into nucleus, which stabilizes c-myc and promotes the transcription of HIF-1 alpha, and the activates MAPK pathway, increases matrix metalloproteinase (MMP) activity and vascular endothelial growth factor (VEGF) expression, which results in the enhancement of cell invasion and migration.

Citation Format: Sai Ma, Yan-Yi Jiang, Li-Fei Wu, Jia-Jie Hao, Yu Zhang, Xin Xu, Yan Cai, Ming-Rong Wang. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 891. doi:10.1158/1538-7445.AM2017-891