# CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence
## 1 Introduction  
Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumors worldwide and has a poor prognosis. Its main attributes include high heterogeneity,elevated invasive and proliferative capacity, chemoresistance, increased vasculature, and a high rate of recurrence. So far, treatment with alkylating/methylating agents, such as temozolomide, has been the most widespread therapeutic approach together with radiotherapy, with modest clinical benefit [1]. Therefore, elucidation of the molecular mechanisms driving tumorigenic potential and invasiveness in GBM is of utmost importance for improving current therapeutic strategies.  
CD44, a type I single-pass transmembrane glycoprotein, is an adhesion molecule and a well-established stem cell marker in various types of tumors. CD44 is encoded by a single gene and is generated by 20 exons in humans, giving rise to a plethora of isoforms due to alternative splicing. Post-translational modifications, such as extensive \(O\)- and \(N\)-glycosylation, add to the complexity of its biology. CD44 can be subjected to sequential proteolytic cleavage events, inducing ectodomain shedding and, after a second cleavage in the transmembrane domain, liberation of its intracellular domain (CD44-ICD), which is translocated to the nucleus and affects transcriptional responses [2]. Stimulation by growth factors and cytokines efficiently activate CD44 cleavage in various types of cancers, such as transforming growth factor \(\beta\) (TGF\(\beta\)) in lung cancer [3] and platelet-derived growth factor (PDGF)-BB in breast cancer [4].  
CD44 binds hyaluron, a constituent of the extracellular matrix (ECM), via the Link domain in its N-terminus, which triggers downstream signaling by promoting the interaction of the CD44 cytoplasmic tail with members of the ERM family and ankyrin [5; 6]. Hyaluronan-CD44 interactions have been shown to be pivotal in various aspects of tumor progression, such as proliferation, migration, invasion, survival, and stemness. During cancer progression, hyaluron synthase 2 (HAS2) and the cell migration inducing hyaluronidase 2 (CEMIP2, also known as TMEM2), are upregulated, promoting tumorigenesis [7; 8]. HAS2 expression is regulated both at the transcriptional and translational levels; for instance, in response to PDGF-BB and phorol 12-myristate 13-acetate (PMA) [9; 10; 11; 12], but also through the long non-coding RNA HAS2-AS1 [13] that is elevated in gliomas [14; 15]. Hyaluronan is an abundant component of the brain extracellular matrix and promotes glioma cell invasion; however, the molecular mechanisms involved are not well understood [16; 17].  
CD44 levels correlate with GBM progression [18] and high levels of CD44 promote invasion of tumor cells [19]. Moreover, treatment with an anti-CD44 monoclonal antibody impeded glioma growth in mice [20]. The binding of CD44 to osteopontin, which is also abundant in brain tumors, leads to enhanced CD44 cleavage and the released CD44-ICD enhances the expression of hypoxia-inducible factor HIF-2\(\alpha\), promoting a hypoxic response in necrotic and perivascular areas [21].  
The aim of this study was to elucidate the mechanisms involved in the pro-tumorigenic effects of CD44 in GBM. Depletion of CD44 in U251 MG glioblastoma cells caused decreased proliferation and stemness, and acquisition of a pro-senescence state. Transcriptomic analysis of U251 MG cells, grown in stem cell-enriched sphere-like conditions, revealed that CD44 is required for the expression of enzymes involved in the synthesis and degradation of hyaluron, PDGF isoforms, and PDGF receptors, as well as for genes related to the reactive oxygen species (ROS) pathway, glycolysis, and progression from the G2 to M phase. Intriguingly, HAS2 knockdown led to decreased CD44 levels and impaired expression of hypoxia-related genes, implying a CD44/hyaluronan double-positive feedback circuit, driving GBM progression.