Significance of serglycin and its binding partners in promoting metastasis of esophageal squamous cell carcinoma

Abstract

Esophageal cancer, of which esophageal squamous cell carcinoma (ESCC) is the predominant subtype in Asia, is a malignant human cancer with a high incidence rate and poor survival rate. The metastasis of ESCC at late stage is one of the major reasons for its high lethality. Unfortunately, the common cancer biomarkers have low sensitivity and specificity in esophageal cancer. Therefore, a better understanding of the molecular mechanisms underlying ESCC progression is needed to identify novel diagnostic markers and therapeutic targets for intervention. Cancer cell invasion, which involves changes in cell morphology and gene expressions, is a crucial step of metastasis. A previous study in our group showed that SRGN (encoding serglycin) was the top upregulated gene in a highly invasive ESCC subline, suggesting that it might have a role in ESCC invasion. SRGN is a proteoglycan with a core protein containing an attachment region for glycosaminoglycan (GAG) side chains. The functional significance of SRGN in several human cancers has been reported in recent years. However, little is known about the diagnostic or prognostic significance of circulating SRGN. Its functions and mechanisms in ESCC progression have not been documented. In this study, stable overexpression of SRGN was found to promote invasion and metastasis of ESCC cells, and the function was dependent on the integrity of its GAG attachment domain. Reverse Phase Protein Array analysis showed that SRGN activated the MAPK/ERK pathway. The activation of the MAPK/ERK pathway further contributed to c-Myc stabilization. Moreover, conditioned medium (CM) from SRGN-overexpressing cells promoted invasion of parental cells. This autocrine effect was partly attributed to SRGN-induced midkine (MDK) which, according to data from chemokine array analysis, was a differentially overexpressed chemokine in the CM from SRGN-overexpressing cells. Immunofluorescent staining, proximate ligation assay and co-immunoprecipitation analysis demonstrated that MDK was a novel binding partner of SRGN. It was bound to the GAG chains of glycosylated SRGN in ESCC cells and remained so after secretion. MDK was also found to bind with CD44, a known SRGN receptor, to form a SRGN/MDK/CD44 complex, which might facilitate autocrine stimulation of MAPK/ERK signaling and maintain c-Myc expression. Moreover, SRGN was found to be able to bind to and upregulate the secretion of matrix metalloproteinase (MMP) 2 and MMP9. Using fluorophore-assisted carbohydrate electrophoresis analysis, the GAG chains that contributed to the interaction between SRGN and its binding partners were found to be mainly decorated with chondroitin sulfate comprising of ∆di-4S and ∆di-6S disaccharides. Clinically, multivariate analysis showed that serum SRGN was an independent prognostic marker in patients with ESCC. Serum MDK was positively correlated with serum SRGN expression and had prognostic significance in patients with Stage III/IV ESCC. In conclusion, serum SRGN is a potential non-invasive biomarker with prognostic significance in ESCC. MDK is a novel GAG-dependent binding partner of SRGN which contributes to ESCC progression by upregulating MAPK/ERK signaling and c-Myc expression. Strategies that target SRGN, its binding partners, or its GAG chains where most of the protein interactions take place, may be a new direction in ESCC therapy.