# CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence
## 3 Results
Proliferation of glioma cells has been correlated to CREB activation [28], and since CD44 signalling is important for sustaining active CREB [29], we analysed the phosphorylation status of S132 in CREB by immunoblotting; dramatically reduced p-CREB levels were detected in CD44 KO clones in comparison to control cells, both in the presence and absence of serum (Figure 1e). Hence, these observations imply an essential role of CD44 in CREB activation and proliferation of U251 MG cells. Because the CD44 KO U251 MG cells showed a reduced rate of proliferation, we investigated the possibility that they had undergone cellular senescence. For this purpose, we used a senescence-associated \(\beta\)-galactosidase (SA-\(\beta\)-gal) assay on cultures of the parental cells or selected CD44 KO clones grown in a medium without or with 5% serum. Interestingly, strong \(\beta\)-gal staining was detected in CD44 KO clones grown in the absence of serum compared to control cells; this effect disappeared by increasing the serum concentration, indicating that the abolition of CD44 signaling causes growth arrest and senescence under challenging culture conditions (Figure 1f). To further corroborate our observations, we monitored levels of the cell cycle inhibitor and well-established senescence marker p16/CDKN2A. Immunoblotting analysis revealed enhanced protein levels of p16 in CD44 KO clones compared to parental cells, an effect that was more prominent in the absence of serum (Figure 1g). Genetic Depletion of CD44 Impairs GBM-Related Gene Signatures and Phenotypes in Sphere-Like Conditions  
In order to address the contribution of CD44 to GBM progression, we performed RNA-sequencing of either U251 MG KO cells (clone #28) or parental cells in the presence or absence of the \(\gamma\)-secretase inhibitor DAPT, which is known to inhibit CD44 cleavage; the CD44 cleavage product CD44-ICD has been implicated in mediating CD44-dependent transcriptional responses [30]. The cells were cultured in anchorage-independent sphere-like conditions. Our transcriptomic analysis revealed that, in total, 1684 genes were differentially expressed upon genetic depletion of CD44; 997 of them were down- and 687 upregulated in the CD44 KO cells compared to the control cells (Figures 2a and S2a and Tables S1-S3). Moreover, treatment with DAPT led to altered expression of 1111 genes, of which 736 were down- and 375 were upregulated compared to control cells (Figures 2a and S2b and Tables S1, S2 and S4). Importantly, 161 genes overlapped between CD44 KO cells and parental cells treated with DAPT (Figures 2a and S2c). Gene set enrichment analysis displayed significant downmodulation in gene signatures, characterizing, for example, hypoxia, the reactive oxygen species (ROS) pathway, the glycolytic pathway, and the G2-M phase cell cycle progression, both in CD44 KO cells and DAPT-treated cells, in comparison to parental cells (Figure 2b,c, Tables S1 and S2). By performing qPCR to validate the results of the RNA-seq assay, hypoxia-related genes VEGFA, LOX, and BCL2 mRNA were found to be downregulated in two CD44 KO clones (#28 and #3) and in parental cells treated with DAPT, compared to untreated parental cells (Figure 2d). Figure 2: CD44 genetic depletion and inhibition of CD44 cleavage leads to common gene signatures in U251 MG cells grown in sphere-like conditions. (**a**) Venn diagram of overlapping down- or upregulated  
genes comparing parental U251 MG cells to either CD44 KO cells or U251 MG cells treated with the \(\gamma\)-secretase inhibitor DAPT; \(p\)-values are computed using Fisher's Exact test. Cells were grown in low-attachment conditions. (**b**) FDR \(q\)-values of specific gene signatures (hallmarks) upon CD44 KO or treatment of parental U251 MG with DAPT, compared to control cells. (**c**) Specific gene set enrichment analyses of the transcriptome between parental U251 MG cells and CD44 KO cells, or parental U251 MG cells treated with DAPT. ES, enrichment score; NES, normalized ES; NOMp, nominal \(p\)-value.