Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-β signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-β signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-β and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-β triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-β receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-β-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-β-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.
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.
Three-dimensional tumorsphere cultures recapitulate the expression of several cancer stem cell (CSC) biomarkers and represent an effective in vitro platform to screen the anti-CSC properties of drugs. Whereas ovarian carcinoma is among the leading causes of death for women, ovarian CSC (OvCSC), a highly malignant subpopulation of ovarian cancer cells, is thought to be responsible for therapy resistance, metastasis, and tumor relapse. Epigallocatechin-3-gallate (EGCG), a diet-derived active polyphenol found in green tea leaves, can suppress ovarian cancer cell proliferation and induce apoptosis. However, its capacity to prevent the acquisition of cancer stemness traits in ovarian malignancies remains unclear. Here, we exploited the in vitro three-dimensional tumorsphere culture model to explore the capacity of EGCG to alter CSC biomarkers expression, signal transducing events and cell chemotaxis. Total RNA and protein lysates were isolated from human ES-2 ovarian cancer cell tumorspheres for gene assessment by RT-qPCR and protein expression by immunoblot. Real-time cell chemotaxis was assessed with xCELLigence. Compared with their parental adherent cells, tumorspheres expressed increased levels of the CSC markers NANOG, SOX2, PROM1, and Fibronectin. EGCG treatment reduced dose-dependently tumorspheres size and inhibited the transcriptional regulation of those genes. Src and JAK/STAT3 signaling pathways appeared to be relevant for CSC phenotype and chemotactic response. In conclusion, these data highlight and support the chemopreventive benefits of the diet-derived EGCG and its capacity to target intracellular transducing events that regulate the acquisition of an invasive CSC phenotype.
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