Ras suppressor-1 (RSU-1) promotes cell invasion in aggressive glioma cells and inhibits it in non-aggressive cells through STAT6 phospho-regulation

Abstract

Most gliomas are invasive tumors formed from glial cells and associated with high mortality rates. In this study, we characterized four glioma cell lines of varying degree of aggressiveness (H4, SW1088, A172 and U87-MG) in terms of morphology, cytoskeleton organization and stiffness, and evaluated their invasive potential by performing invasion, colony forming and spheroid invasion assays. Cells were divided into two distinct groups: aggressive cell lines (A172 and U87-MG) with more elongated, softer and highly invasive cells and less aggressive cells (H4 and SW088). Interestingly, we found that Ras Suppressor-1 (RSU-1), a cell-matrix adhesion protein involved in cancer cell invasion, was significantly upregulated in more aggressive glioma cells compared to less aggressive. Importantly, RSU-1 silencing had opposing effects on glioma cell invasion depending on their aggressiveness, inhibiting migration and invasion of aggressive cells and promoting those of less aggressive cells. Finally, we found that RSU-1 silencing in aggressive cells led to decreased Signal Transducer and Activator of Transcription6 (STAT6) phosphorylation and Matrix Metalloproteinase13 (MMP13) expression in contrast to less invasive cells. Our study demonstrates that RSU-1 promotes invasion of aggressive glioma cells and inhibits it in the non-aggressive cells, indicating that it could serve as a predictor of gliomas progression.

Coordinated Expression of Ras Suppressor 1 (RSU-1) and Growth Differentiation Factor 15 (GDF15) Affects Glioma Cell Invasion

“…Since the role of GDF15 in cancer progression is controversial and not fully elucidated yet [50,51], we first tested GDF15 expression in three cell lines H4, SW1088, and A172 both at the mRNA (Figure 1A) and protein level (Figure 1B,C). In our previous work [34], we have shown that A172 cells that cause GBM are very aggressive having a strong invasive capacity in contrast to SW1088 cells, which cause astrocytoma and are less invasive, and H4 cells which are almost non-invasive neuroglioma cells. Here, we show that H4 cells express GDF15 at higher levels than SW1088 and A172 cells both at the mRNA and protein level (Figure 1), whereas RSU-1 expression follows the exact opposite pattern, being elevated as the aggressiveness of cells increases (Figure 1D–F).…”

“…RSU-1, which binds to PINCH1 at FA sites [19] has been previously found to be upregulated in more invasive breast and brain cancer cells [29,34]. Moreover, we recently demonstrated a differential regulation of cell migration and invasion of glioma cells by RSU-1 based on their aggressiveness, with RSU-1 promoting an invasive behavior in aggressive cells (A172 and U87-MG) and inhibiting them in the less aggressive ones (H4 and SW1088) [34]. This by itself indicates the existence of a complex molecular mechanism that governs glioma cell invasion in vitro.…”

“…To investigate the interplay and possible connection between RSU-1 and GDF15 in glioma cells, we used three different brain cell lines, namely H4, SW1088 and A172, which have different tumoral origin, properties and proteins expression level [34,56,57]. and four different experimental approaches; (a) hrGDF15 treatment, (b) RSU-1 silencing, (c) GDF15 silencing, and (d) combined hrGDF15 treatment and RSU-1 silencing.…”

“…Regarding the role of RSU-1 in the central nervous system [31,32,33], not much is known either. Interestingly though, our recent work demonstrated a differential regulation of cell migration and invasion of glioma cells by RSU-1 based on their aggressiveness [34]. Thus, RSU-1 was shown to promote the invasion capacity of aggressive glioma cells (A172 and U87-MG) but inhibit that of non-aggressive cells (H4 and SW1088), indicating that a complex molecular mechanism is in place.…”

Hertz model or Oliver & Pharr analysis? Tutorial regarding AFM nanoindentation experiments on biological samples

“…Briefly, before each experiment, the cells were washed twice with PBS and new complete media was added. The petri dish (35 mm) was mounted directly on the AFM specimen disc and all the experiments were performed under liquid condition (cells' complete media) at room temperature [10,41,42]. AFM experiments: A Molecular Imaging-Agilent PicoPlus AFM system (now known as AFM 5500 Keysight technologies) with a round (2.5 μm radius) ball-shape tip (CP-PNPL-BSG), and spring constant of 0.08 N m −1…”