A Signaling Crosstalk Links SNAIL to the 37/67 kDa Laminin-1 Receptor Ribosomal Protein SA and Regulates the Acquisition of a Cancer Stem Cell Molecular Signature in U87 Glioblastoma Neurospheres

Abstract

Background: Three-dimensional in vitro neurospheres cultures recapitulate stemness features associated with poor clinical outcome in glioblastoma patients. They are commonly used to address brain cancer stem cell (CSC) signal transducing biology that regulates spheroids formation and stemness phenotype, and to assess the in vitro pharmacological impact of chemotherapeutic drugs. Objective: Here, we addressed the role of a new signaling axis involved in the regulation of in vitro spheroids formation and assessed the chemopreventive ability of diet-derived epigallocatechin gallate (EGCG) to impact the processes that govern the acquisition of spheroids CSC stemness traits. Methods: Neurospheres were generated from adherent human U87 glioblastoma cancer cell cultures under conditions that recapitulate stemness features. Total RNA and protein lysates were isolated for gene expression by RT-qPCR and protein expression by immunoblot. Transcriptomic analysis was performed through RNA-Seq. Results: Compared to their parental adherent cells, tumorspheres expressed increased levels of the CSC markers NANOG, SOX2, PROM1 (CD133), as well as of the epithelial-to-mesenchymal transition (EMT) markers Fibronectin, SNAI1, and 37/67 kDa laminin-1 receptor ribosomal protein SA (RPSA). Increased PROM1, SOX2, Fibronectin, and RPSA transcripts level were also observed in clinical grade IV glioblastoma tissues compared to normal tissue. EGCG treatment reduced dose-dependently tumorspheres size and inhibited the transcriptional regulation of those genes. An apoptotic signature was also found in spheroids with increased signal transducing events involving GSK3α/β, RSK, and CREB. These were repressed upon RPSA gene silencing and partially by SNAI1 silencing. Conclusion: This work highlights a signaling axis linking RPSA upstream of SNAIL in neurospheres genesis and supports the chemopreventive impact that diet-derived EGCG may exert on the acquisition of CSC traits.

A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype

“…Neurospheres culture conditions are known to recapitulate, in part, the cancer stem cells (CSC) phenotype ( 36 , 39 ). U87 glioblastoma 3D neurosphere cultures appeared to reach maturation at 72 hours ( Figure 5A ) as described previously ( 40 ). To address how such phenotype impacted the G6PC3 , SLC37A2 , and SLC37A4 transcript levels, total RNA was isolated, and qPCR analysis was performed in comparison to 2D cell monolayers.…”

“…Notably, the expression levels of G6PC3 and SLC37A4 were found induced in neurospheres, a 3D cell culture model which recapitulates a stem cell-like phenotype associated with increased tumorigenicity and therapeutic resistance ( 36 , 39 , 40 ). This observation suggests that transcriptional manipulation of G6PC3 and SLC37A4 levels may contribute to the chemoresistance and invasive molecular signature of GBM and of GBM-derived CSC.…”

Epigallocatechin-3-Gallate Prevents the Acquisition of a Cancer Stem Cell Phenotype in Ovarian Cancer Tumorspheres through the Inhibition of Src/JAK/STAT3 Signaling

“…Celprogen provided human ovarian cancer stem cells (OvCSC) (San Pedro, CA, USA). Cells were cultivated as monolayers at 37 °C in a humidified atmosphere (5% CO 2 ) using the appropriate expansion and undifferentiation media, as well as matrix pre-coated flasks, according to the manufacturer’s recommendations (Celprogen).ES-2 tumorsphere formation was adapted from established protocols [ 20 , 21 , 22 , 23 , 24 ] and performed as follows: 80–90% adherent ES-2 monolayer cells were trypsinized and plated in non-adherent bacterial dishes at a density of 2 × 10 5 cells/mL in complete media for 24 h. Following that, the supernatant was removed and serum-free McCoy’s 5a Modified Medium was supplemented with 10 ng/mL human basic fibroblast growth factor (Gibco, Thermo Fisher, 13256029), 20 ng/mL human epidermal growth factor (Gibco, Thermo Fisher, PHG0315), 5 μg/mL insulin (Sigma Aldrich, I3536), and bovine serum albumin (BSA) (Sigma Aldrich, A9418-5G) at 4% was carefully added to the dishes. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .…”

Correction: Gresseau et al. A Signaling Crosstalk Links SNAIL to the 37/67 kDa Laminin-1 Receptor Ribosomal Protein SA and Regulates the Acquisition of a Cancer Stem Cell Molecular Signature in U87 Glioblastoma Neurospheres. Cancers 2022, 14, 5944

“…In the original publication [ 1 ], there was an honest mistake in Figure 4D (P-AKT panel) as published. The P-AKT expression was erroneously duplicated from Figure 5D.…”