# CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence
## 4 Discussion  
The aim of the present study was to shed light on the molecular events driving CD44-dependent GBM aggressiveness [34]. In line with the vast body of evidence implicating CD44 in glioma progression [16], we observed a significant reduction in the proliferative capacity of the well-established GBM in vitro model U251 MG, upon depletion of CD44 by CRISPR/Cas9 gene editing. In the case of thyroid cancer cells, CD44 controls cell cycle progression and proliferation via sustaining phosphorylation and activation of the CREB transcription factor. CD44 cleavage and the liberated CD44-ICD seem to be required for this event [29]. Importantly, activation of CREB has been shown to be prominent and indispensable for proliferation of glioma tumors [28]. In agreement with these studies, we noticed a robust decrease in p-CREB levels in the presence or absence of serum in the CD44-deprived cells (Figure 1e), suggesting that activation of CREB downstream of CD44 is required to drive proliferation of U251 MG cells. Prolonged cell cycle arrest triggers senescence. We noticed that challenging cells by deprivation of serum in the culture medium caused upregulation of the cell cycle inhibitor p16/CDKN2A, promoting cellular senescence of CD44 KO cells. Notably, expression of  
Figure 5: HAS2 downregulation impairs CD44-related genes and self-renewal capacity. (**a**) U251 MG cells stably transfected with shRNAs against _HAS2_ or control shRNA were grown in sphere-like conditions. _HAS2-AS1_, _HAS2_, _VEGFA_, and _LOX_ mRNAs were quantified by real-time qPCR analysis and normalized to _HPRT_. (**b**) An ELDA assay was carried out in the _HAS2_ knockdown and control cells, and cells treated with the hyaluron synthesis inhibitor 4-MU or DAPT. One representative experiment out of two is depicted. CD44 has been inversely correlated with expression of p16, as well as p53, both having pivotal roles in senescence acquisition [35]. To delineate the role of CD44 in GBM progression, we performed RNA-seq analysis upon genetic depletion of CD44 or inhibition of its cleavage by the well-established \(\gamma\)-secretase inhibitor DAPT. Our analysis revealed a CD44-dependency for gene signatures related to hypoxia, glycolysis, ROS pathway, and progression from the G2 to M cell cycle phase. Interestingly, treatment with DAPT also affected similar pathways, indicating that at least a part of the CD44-dependent transcriptional responses is dependent on the generation of its cleavage product CD44-ICD. Our findings are consistent with a previous report showing that CD44 enhances the glycolytic pathway during cancer progression [36]. In cells grown in stem-cell enriching sphere-like conditions, the expression of molecules involved in synthesis and degradation of hyaluron and members of the PDGF and PDGF receptor families were found to be dependent on CD44 expression. Importantly, _PDGFRA_ (17.3%) and _KIT_ (11.7%) have been found to be the most amplified genes encoding receptor tyrosine kinases (RTK) after _EGFR_ (56%) in a cohort of more than 500 GBM tumors [37]. _PDGFRA_ was strongly downregulated in CD44-depleted cells; concurrently, _PDGFB_ and _PDGFD_, generating the homo-dimeric ligands PDGF-BB and PDGF-DD [38], were suppressed upon CD44 loss. Therefore, it is plausible that autocrine PDGF signaling via both PDGFRA and PDGFRB is impaired in U251 MG cells upon CD44 KO. Our results also revealed that KIT expression was severely diminished upon CD44 loss or after inhibiting its cleavage, indicating that _KIT_ may be a CD44-ICD target gene. Intriguingly, the KIT ligand SCF potentially induced CD44 cleavage, suggesting a positive regulatory feedback loop between CD44 and SCF/KIT signaling. Noteworthy, we observed reduced stem cell frequency in CD44 KO cells compared to the parental U251 MG, as shown by ELDA (Figure 2e). Our transcriptomic analysis revealed downregulation of hypoxia-related genes in CD44 KO cells compared to the parental cells.