Role of Id1 in the regulation of esophageal tumor micro- and macroenvironment

Abstract

Esophageal cancer is the 8th most common cancer worldwide and esophageal squamous cell carcinoma (ESCC) is the predominant form. Despite the advances in surgical techniques and chemoradiotherapy, the 5-year survival rate of patients remains poor. Cancer has been described as a systemic disease. Tumor cells not only interact with the stroma in the local environment (tumor microenvironment), but also influence and respond to the host systemic environment (tumor macroenvironment). However, our understanding of the underlying cellular and molecular mechanisms is still inadequate. It was reported that the majority of ESCC overexpress Id1 (inhibitor of differentiation or DNA binding). The previous studies in our laboratory showed that Id1 promotes tumor growth and metastasis through activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway, and that this activation is mediated by Id1-induced secretion of insulin-like growth factor 2 (IGF2) from the cancer cells.

For the first time, the results of the present study showed that Id1-induced IGF2 also exerted paracrine effects on fibroblasts, which comprise a prominent cell type in the tumor microenvironment, and activated them to produce vascular endothelial growth factor (VEGF). Data from Western blot and ELISA showed that the addition of conditioned medium from the Id1-overexpressing ESCC cells or recombinant human IGF2 not only instigated esophageal fibroblasts to produce VEGF, but also enhanced their migratory potential, and that the effect was attenuated by IGF2-knockdown in cancer cells, or addition of IGF2-neutralizing antibody to the conditioned medium. In addition, the IGF2-activated fibroblasts were found to exert multifaceted roles in the tumor microenvironment.

Through a combination of in silico prediction and TaqMan miRNA assay, miR-127-5p and miR-29c were identified as potential mediators in the regulation of IGF2 on VEGF. Subsequent gain- and loss-of-function experiments, as well as luciferase assays, confirmed that miR-29c bound directly to the 3’UTR of VEGF to mediate the regulation of IGF2 on VEGF. Next, p53 was demonstrated to play a crucial role in mediating the regulation of IGF2 on the expression of miR-29c by interacting directly with the promoter of miR-29c.

Subsequently, tumor xenograft experiments using nude mice that had received bone marrow transplantation from green fluorescent protein (GFP) transgenic mice showed that Id1-expressing ESCC tumor could exert a systemic effect on the tumor macroenvironment by instigating the incorporation of VEGFR1-positive bone marrow cells into primary tumors and secondary sites to facilitate tumor growth and formation of the pre-metastatic niche. In addition, bone marrow cells from mice bearing Id1-expressing tumor xenografts directly facilitated tumor growth and distant metastatic colonization, whereas systemic administration of VEGFR1 antibody significantly abrogated these effects. Moreover, the CXCL5/CXCR2 axis was found to significantly contribute to the formation of pre-metastatic niche in lungs.

Furthermore, preclinical data from this study showed for the first time that targeting host VEGFR1 and VEGFR2 using neutralizing antibodies could reduce host 〖VEGFR1〗^+/〖VEGFR2〗^+ cells and suppress angiogenesis, esophageal cancer growth and metastasis. Taken together, this study demonstrated a novel paradigm of ESCC progression that involves the orchestration of cancer and non-cancer cells in the tumor micro- and macroenvironments by the Id1/IGF2/VEGF/VEGFR1 cascade.