QSOX1 Modulates Glioblastoma Cell Proliferation and Migration In Vitro and Invasion In Vivo.

Simple Summary: Glioblastoma (GBM) is the deadliest form of primary brain cancer, and the survival of patients is only about 15 months after diagnosis, even with aggressive treatment. Many cellular factors that are used by GBM cells to divide abnormally and invade brain tissue to result in patient death are unknown. Here, we investigated the potential role of the cellular enzyme QSOX1, which creates specific bonds within proteins, in GBM cell proliferation, migration in a dish, and invasion into brain tissue in our chick embryo brain tumor model system. By experimentally reducing QSOX1 protein production in GBM cells, we found that this reduction resulted in less proliferation, slower migration, and less invasion into brain tissue. These results show the importance of the QSOX1 enzyme in GBM cells in order for them to exhibit their abnormal aggressive behavior that drives this incurable cancer. Background: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme that catalyzes the oxidation of free thiols to generate disulfide bonds in a variety of proteins, including the cell surface and extracellular matrix. QSOX1 has been reported to be upregulated in a number of cancers, and the overexpression of QSOX1 has been correlated with aggressive cancers and poor patient prognosis. Glioblastoma (GBM) brain cancer has been practically impossible to treat effectively, with cells that rapidly invade normal brain tissue and escape surgery and other treatment. Thus, there is a crucial need to understand the multiple mechanisms that facilitate GBM cell invasion and to determine if QSOX1 is involved. Methods and Results: Here, we investigated the function of QSOX1 in human glioblastoma cells using two cell lines derived from T98G cells, whose proliferation, motility, and invasiveness has been shown by us to be dependent on disulfide bond-containing adhesion and receptor proteins, such as L1CAM and the FGFR. We lentivirally introduced shRNA to attenuate the QSOX1 protein expression in one cell line, and a Western blot analysis confirmed the decreased QSOX1 expression. A DNA content/cell cycle analysis using flow cytometry revealed 27% fewer knockdown cells in the S-phase of the cell cycle, indicating a reduced proliferation. A cell motility analysis utilizing our highly quantitative SuperScratch time-lapse microscopy assay revealed that knockdown cells migrated more slowly, with a 45% decrease in migration velocity. Motility was partly rescued by the co-culture of knockdown cells with control cells, indicating a paracrine effect. Surprisingly, knockdown cells exhibited increased motility when assayed using a Transwell migration assay. Our novel chick embryo orthotopic xenograft model was used to assess the in vivo invasiveness of knockdown vs. control cells, and tumors developed from both cell types. However, fewer invasive knockdown cells were observed after about a week. Conclusions: Our results indicate that an experimental reduction in QSOX1 expression in GBM cells leads to decreased cell proliferation, altered in vitro migration, and decreased in vivo invasion. [ABSTRACT FROM AUTHOR]