Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions

Abstract

Suprabasin (SBSN) is a secreted protein that is isolated as a novel gene expressed in differentiated keratinocytes in mice and humans. It induces various cellular processes such as proliferation, invasion, metastasis, migration, angiogenesis, apoptosis, therapy and immune resistance. The role of SBSN was investigated in oral squamous cell carcinoma (OSCC) under hypoxic conditions using the SAS, HSC-3, and HSC-4 cell lines. Hypoxia induced SBSN mRNA and protein expression in OSCC cells and normal human epidermal keratinocytes (NHEKs), and this was most prominent in SAS cells. The function of SBSN in SAS cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); 5-bromo-2'-deoxyuridine (BrdU); cell cycle, caspase 3/7, invasion, migration, and tube formation assays; and gelatin zymography. Overexpression of SBSN decreased MTT activity, but the results of BrdU and cell cycle assays indicated upregulation of cell proliferation. Western blot analysis for cyclin-related proteins indicated involvement of cyclin pathways. However, SBSN did not strongly suppress apoptosis and autophagy, as revealed by caspase 3/7 assay and western blotting for p62 and LC3. Additionally, SBSN increased cell invasion more under hypoxia than under normoxia, and this resulted from increased cell migration, not from matrix metalloprotease activity or epithelial-mesenchymal transition. Furthermore, SBSN induced angiogenesis more strongly under hypoxia than under normoxia. Analysis using reverse transcription-quantitative PCR showed that vascular endothelial growth factor (VEGF) mRNA was not altered by the knockdown or overexpression of SBSN VEGF, suggesting that VEGF is not located downstream of SBSN. These results