Role of Id1-IGF2-VEGF-VEGFR relay in esophageal tumorigenesis

Abstract

INTRODUCTION: It is increasingly evident that the close interactions between cancer cells and other cells in the tumor microenvironment help drive tumor progression. Esophageal squamous cell carcinoma (ESCC) is a highly lethal disease, and there is an association between poor clinical outcome and Id1 overexpression in ESCC. We previously reported that Id1 induces the expression and secretion of insulin-like growth factor-2 (IGF2) which promotes esophageal cancer progression and metastasis in an autocrine manner. In this study, we aim to investigate the paracrine effect of Id1-induced IGF2 on esophageal fibroblasts, and the stimulatory effects and roles of fibroblast-secreted vascular endothelial growth factor (VEGF) on endothelial cells and VEGF receptor (VEGFR)-positive bone marrow cells during esophageal tumorigenesis. MATERIALS AND METHODS: Human ESCC cell lines with ectopic expression of Id1, co-expression of Id1 and shRNA against IGF2 (shIGF2), or empty vector were established. Bone marrow was harvested from femurs and tibias of nude mice for isolation of VEGFR1+ bone marrow cells by flow cytometry. Cell proliferation and migration were determined using MTT and chamber migration assays respectively. Western blot and ELISA were used to determine the protein expression and concentration in cell lysates and conditioned medium (CM). Tube formation assay was used to analyze the in vitro angiogenic ability of endothelial cells. Human ESCC cells were subcutaneously injected into the flanks of nude mice to establish the tumor xenografts. RESULTS AND DISCUSSION: Our data from Western blot and ELISA showed that the addition of CM from the ESCC cells with overexpression of Id1 or recombinant human IGF2 alone could promote esophageal fibroblasts to produce VEGF in vitro, and co-expression of shIGF2 or neutralizing antibody against IGF2 greatly diminished this effect. We also found that the CM collected from IGF2-activated fibroblasts could enhance the proliferation, tube-formation and migration abilities of human umbilical vein endothelial cells (HUVECs). The addition of VEGF-neutralizing antibody effectively attenuated these stimulatory effects. In addition, our results showed that the CM collected from IGF2-activated fibroblasts increased the mobility of VEGFR1+ bone marrow cells. Furthermore, data from in vivo experiment showed that bone marrow cells harvested from nude mice bearing Id1-expressing tumor xenografts had tumor-promoting activity when co-injected subcutaneously with untransfected ESCC cells into new groups of nude mice. CONCLUSIONS: Id1-overexpressing ESCC cells secrete IGF2 and educate stromal fibroblasts to secrete VEGF, which may in turn activate and mobilize endothelial cells as well as bone marrow-derived hematopoietic-progenitor cells to promote tumor angiogenesis.