Small Integrin-Binding N-Linked Glycoproteins (SIBLINGS) are upregulated in human salivary gland cancer

Abstract

Introduction: Salivary gland carcinomas constitute a rare but deadly group of head and neck cancers, but timely diagnosis is often delayed due to inherent variability in etiology, heterogeneity and histopathological characterization. SIBLINGs are a family of secreted glycophosphoproteins that include osteopontin (OPN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and matrix extracellular phosphoglycoprotein (MEPE). SIBLINGs were first discovered in bone and teeth, and were considered to be exclusively expressed in mineralized tissue. In addition to mineralized tissue, SIBLINGs have now been shown to have variable expression in normal, non-mineralized tissue and in cancers. However, there have been no studies evaluating SIBLING expression in human salivary gland cancers. Our study tested the hypothesis that SIBLINGs, specifically, BSP, DSPP and OPN, would be significantly overexpressed in human salivary gland cancer. We also hypothesized that the cancer secretome would influence SIBLING expression in normal salivary gland cells. Methods: Normal and cancerous human salivary gland tissue obtained from the NDRI were processed using routine immunohistochemistry techniques to evaluate expression of BSP, DSP, and OPN. In addition normal HSG cell line and cancer HTB-41 cell line were evaluated using immunofluorescence techniques to localize expression of BSP, DSP and OPN. Normal HSG, cancer HTB-41 and HSG* cells (normal HSG cells exposed to a cancer HTB-41 secretome) were propagated using routine cell culture techniques for 24, 48, and 72 hours. Western blotting techniques were utilized to quantify and compare SIBLING protein expression levels in HSG, HTB-41 and HSG* cells. Normal HSG, cancer HTB-41, and HSG* cells were processed via immunoflourescence in order to observe localization of SIBLINGs. Results: Immunohistochemistry and western blot showed increased expression of SIBLINGs in human salivary gland cancers. Furthermore, immunoflourescence revealed distinct localization of SIBLING proteins in HSG and HTB-41 cell lines. In terms of HSG*, it was found that cells exposed to cancer secretome exhibited similar SIBLING expression to HTB-41. Conclusion: Our studies confirm that SIBLING proteins are selectively expressed in human salivary gland cancer. Also, the cancer secretome is found to affect SIBLING expression in normal cells, similar to HTB-41 cancer cell lines.