Interleukin-1Beta and exosomal M6PR secreted by serglycin-overexpressing esophageal cancer cells instigate fibroblasts and endothelial cells to promote esophageal cancer progression

Abstract

Non-tumor cells can be recruited and educated by cancer cells to facilitate cancer progression. Previously, we found that serum serglycin (SRGN), a secretory proteoglycan, was an independent prognostic marker for patients with esophageal squamous cell carcinoma (ESCC), and that the autocrine pro-invasive effect of SRGN on ESCC cells was mediated by midkine (MDK). Here, we investigated the effects of cancer cell-derived SRGN on human esophageal fibroblasts (HEF) and human umbilical vein endothelial cells (HUVECs). We found that conditioned medium from SRGN-overexpressing ESCC cells (SRGN-CM) promoted the migration and proliferation of HEF. After SRGN-CM treatment, HEF showed increased expression of fibroblast activation protein alpha (FAP), hepatocyte growth factor (HGF) and amphiregulin, and could enhance tumor growth in vivo. In addition, exosomes derived from SRGN-overexpressing ESCC cells (SRGN-Exo) enhanced the tube formation ability of HUVECs. We found that the effects of SRGN-CM on activation, migration and proliferation of HEF were mediated by MDK. To elucidate the mechanisms by which SRGN upregulates HGF and amphiregulin, and promotes endothelial tube formation, cytokine array and mass spectrometry were performed to analyze differentially expressed proteins in SRGN-CM and SRGN-Exo respectively. The results showed upregulated secretion of interleukin (IL)-1β, IL-18 and tumor necrosis factor-α in SRGN-CM, as well as enriched cation-dependent mannose-6-phosphate receptor (M6PR), integrin alpha-5, teneurin-2 and neurogenic locus notch homolog protein 2 in SRGN-Exo, which were validated by Western blot. These effects were dependent on the glycosaminoglycan chains on SRGN. Our data also showed that the enhanced secretion of IL-1β promoted the expression of HGF in HEF by activating extracellular signal-regulated kinase/activating protein-1. Treatment with SU11274, a c-Met (the receptor of HGF) inhibitor, attenuated the proliferation of ESCC cells co-cultured with HEF, which further indicates that IL-1β-induced HGF from HEF plays a significant role in the tumor microenvironment. In addition, the upregulated exosomal M6PR was found to mediate the enhancing effect of SRGN-Exo on endothelial tube formation ability. Notably, the expression level of M6PR in serum samples of patients with ESCC was positively correlated with that of SRGN and with poor survival. Taken together, SRGN overexpression in ESCC cells created a tumor-promoting microenvironment by altering the ESCC cell secretome including exosomes to exert influence on HEF and HUVECs. [This study was supported by Research Grants Council of the Hong Kong SAR, China, GRF Project No. 17100819]