CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence

SIMPLE SUMMARY: The hyaluronan receptor CD44 has an important role in glioblastoma multiforme (GBM) progression, but the precise mechanisms have not been elucidated. We have analyzed U251MG glioma cells, expressing CD44 or not, and grown in stem cell-like enriched spheres. Our results revealed that CD44 is important for cell growth and stemness, and for the prevention of senescence. Analysis by RNA sequencing revealed that CD44 is important for the interaction with the hyaluronan-enriched microenvironment. In addition, CD44 depletion impairs certain gene signatures, such as those for platelet-derived growth factor (PDGF) isoforms and PDGF receptors, as well as signatures related to hypoxia, glycolysis, and anti-tumor immune responses. ABSTRACT: Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the γ-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.