Importance of Cholesterol-Enriched Domains and Flotillin-1 in Glioblastoma Cells Invasion

Details

Supervisors

Tyteca, Donatienne

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

enGlioblastoma (GBM) is the most common and aggressive malignant brain tumor, characterized by high invasiveness and therapy resistance. A better understanding of its development and progression mechanisms is therefore required to find new therapies. This master thesis started following the observation from the host laboratory that four GBM cell lines (i.e. U87, U251, SNB19 and LN229) exhibited distinct cholesterol (chol) content and extractability by methyl-β-cyclodextrin (mβCD). My objective was therefore to evaluate whether and how chol and chol-associated protein distributions are altered in GBM cell lines and how these alterations could affect cell migration and invasion in 2D cell lines and 3D spheroids. We first compared the GBM cell lines for storage and surface distribution of chol as well as for the expression and distribution of three chol-associated proteins, i.e. Flotillin-1 (FLOT1), Caveolin-1 (CAV1) and CAVIN1. U87 and LN229, which presented a similar low chol content but distinct extractability by mβCD, also exhibited distinct repartition of chol-enriched domains between the extracellular matrix (ECM)-free and contact sides. FLOT1 was predominantly intracellular in all GBM cell lines, with proximity to CAV1 observed in SNB19 and U251. In contrast, CAV1 and CAVIN1 showed different subcellular/submembrane localization in the four cell lines and were mostly colocalized in U87 and LN229. All those data suggested different types of chol-enriched regions in GBM cells. To assess the functional relevance of these distinct domains, we tested whether surface chol depletion combined or not with FLOT1 or CAV1 knockdown could impact migration and invasion of GBM cells in 2D and 3D spheroids. The results revealed no effect of mβCD on 2D migration and invasion but a modest reduction of 3D spheroid invasion. CAV1 knockdown decreased invasion only in U251. Conversely, knockdown of FLOT1 strongly reduced invasion in all spheroids except U87. Moreover, when combined with mβCD, FLOT1 induced a decrease of invasion across all four GBM cell lines, including U87. Those results were discussed in the context of GBM cell line mechanisms of invasion, i.e. nuclear deformation, ECM degradation and invadopodia formation. Altogether, these findings indicate that chol-enriched domains and FLOT1 represent two complementary targets to decrease cell invasion in GBM.