Role of serglycin in the tumor microenvironment of esophageal squamous cell carcinoma

Abstract

It is well known that cancer cells can recruit and influence non-tumor cells in the tumor microenvironment to provide a tumor-supporting milieu. A previous study showed that serglycin (SRGN), a secretory proteoglycan, can facilitate invasion and metastasis of esophageal squamous cell carcinoma (ESCC) cells in an autocrine manner by augmenting the secretion of midkine (MDK). Here, I investigated the reciprocal interactions between SRGN-overexpressing ESCC cells and non-tumor cells (i.e., fibroblasts and endothelial cells) in the tumor microenvironment. The results showed that SRGN-induced MDK facilitated fibroblast activation protein-α (FAP) upregulation, proliferation and migration of fibroblasts. KEGG pathway analysis showed that calcium and cAMP signaling pathways, which are related to fibroblast transformation, were enriched in MDK-treated fibroblasts. Immunohistochemical analysis of ESCC tissue microarrays showed a positive correlation between SRGN in cancer cells and FAP in stromal cells. SRGN overexpression in ESCC cells also led to increased secretion of interleukin (IL)-1β, tumor necrosis factor-α and IL-18 from these cells. Except for IL-18, this effect was dependent on the glycosaminoglycan side chains of SRGN. Moreover, SRGN-induced IL-1β promoted the expression of hepatocyte growth factor (HGF) and amphiregulin in fibroblasts by activating the phospholipase C γ 1/ extracellular signal-regulated kinase 2/activating protein-1 (AP-1) signaling pathway. Both inhibition of AP-1 with T-5224 and knockdown of c-Fos attenuated the upregulation of HGF and amphiregulin. Positive correlations were found between mRNA levels of SRGN and FAP, SRGN and IL-1β, SRGN and HGF, and between IL-1β and amphiregulin in The Cancer Genome Atlas esophageal carcinoma dataset. In addition, ESCC cells mixed with fibroblasts pretreated with CM from SRGN-overexpressing ESCC cells exhibited increased tumorigenicity in vivo. Results from endothelial tube formation assays suggested that fibroblast-derived HGF was instrumental in promoting angiogenesis in SRGN-overexpressing tumors.

Accumulating evidence shows that exosomes play an essential role in invasion, metastasis of cancer cells and tumor vascularization by transferring functional RNAs, lipids and proteins between different cell types. However, the functions of exosomal proteins secreted by cancer cells were relatively less studied. Therefore, I investigated the effects of exosomes from SRGN-overexpressing ESCC cells (SRGN Exo) on ESCC cells and endothelial cells. The results showed that SRGN Exo facilitated the invasion of ESCC cells and tube formation of endothelial cells in vitro. SRGN Exo were then analyzed by mass spectrometry and Western blot. The results showed that cation-dependent mannose-6-phosphate receptor (M6PR) and ephrin type-B receptor 4 (EphB4) were pronouncedly upregulated in SRGN Exo. Upregulated exosomal M6PR mediated the pro-angiogenic effects of SRGN Exo both in vitro and in vivo. Moreover, there was a positive correlation between SRGN and M6PR in the serum of ESCC patients. High levels of serum M6PR were associated with poor overall survival rates. Data from Gene Expression Omnibus database showed that M6PR was overexpressed in ESCC. In vitro studies showed that M6PR manipulation affected the viability and migration of ESCC cells.

Taken together, SRGN overexpression in ESCC cells creates a tumor-promoting microenvironment by altering the ESCC cell secretome including exosomes to exert influence on esophageal fibroblasts and endothelial cells.