# CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence
## 3 Results
### CD44 Ablation in U251 MG Cells Down-Regulates Expression of PDGF Family Members  
To investigate the molecular mechanisms by which CD44 contributes to GBM progression, we focused on specific genes being affected by CD44 depletion in our transcriptomic analysis. The results of RNA sequencing revealed that the expression of members of the PDGF and PDGF receptor families, and particularly _KIT_, _PDGFRA_, _PDGFB_, and _PDGFD_, were significantly suppressed (Figure 3a). In particular, _KIT_ was one of the most down-regulated genes in the CD44 KO in comparison to parental cells (log2 FoldChange \(-7.38\), FDR = \(1.31\times 10^{-48}\) (Figures 3a and S2a and Table S3), and was found to be downregulated by DAPT treatment as well (log2 FoldChange \(-1.87\), FDR = \(8.64\times 10^{-30}\)) (Figures 3a and S2b and Table S4). Downregulation of _KIT_, _PDGFRA_, _PDGFB_, and _PDGFD_ mRNAs was validated by real-time qPCR analysis in CD44 KO Clones #28 and #3. Interestingly, the expression of _KIT_, but not of _PDGFRA_, was suppressed by treatment with DAPT, suggesting that KIT may be a CD44-ICD target gene. On the contrary, _PDGFB_ and _PDGFD_ were upregulated in the presence of DAPT (Figure 3b). These results were further corroborated at the protein level by immunoblotting for KIT and PDGFRA (Figure 3c). Intriguingly, screening for CD44 cleavage after treatment with several growth factors and cytokines revealed that the KIT ligand, stem cell factor (SCF), more efficiently induced shedding of the ectodomain of CD44 than ligands for other tyrosine kinase receptors (Figure S3). Figure 3: CD44 ablation impairs autocrine PDGF signaling in U251 MG cells grown in sphere-like conditions. (**a**) Volcano plots exhibiting differentially regulated genes, which were either down- or  
upregulated (depicted with blue and red color, respectively) in CD44 KO cells or parental U251 MG cells treated with the \(\gamma\)-secretase inhibitor DAPT, compared to the untreated cells. (**b**) mRNA levels of _KIT_, _PDGFRA_, _PDGFRB_, and _PDGFD_ determined by RT-qPCR in spheres formed by parental U251 MG cells treated or not with DAPT, and CD44 KO cells, are shown after normalization to _TBP_. (**c**) Immunoblotting analysis of PDGFRA, KIT, and CD44 was performed using lysates from U251 MG cells. GAPDH was used as the loading control. Uncropped immunoblots are depicted in Figure S9. Immunoreactive bands were quantified after normalization to GAPDH after using the software ImageJ. Cells were grown in low-attachment conditions in the presence or absence of the \(\gamma\)-secretase inhibitor DAPT. All graphs illustrate the average \(\pm\) SEM values from at least three independent experiments. Asterisks show significant differences compared to the respective control condition: *_p_ < 0.05, **_p_ < 0.01, ***_p_ < 0.001. CD44 Promotes the Synthesis of Hyaluronan in U251 MG Cells and Their Adhesion to the Hyaluronan-Coated Substratum  
Given that hyaluron is a major component of the GBM stroma, we analyzed the impact of CD44 KO on hyaluron synthesis and secretion (Figure 4). RNA-sequencing analysis revealed differences between CD44 KO and parental cells in the expression of mRNAs of molecules involved in hyaluron synthesis, degradation, and its linkage to ECM proteoglycans. In particular, the _HAS2_ levels dropped sharply (log2 FoldChange \(-\)3.18, FDR = 8.9 \(\times\) 10\({}^{-207}\)), as did the levels of its natural antisense transcript _HAS2-AS1_ (log2 FoldChange \(-\)2.11, FDR = 4.45 \(\times\) 10\({}^{-9}\)), which has been shown to be important for its expression [13]. Furthermore, _TMEM2_, encoding a cell surface-localized hyaluronidase, and _VCAN_, encoding the hyaladherin version [31], were also decreased (log2 FoldChange \(-\)1.68, FDR = 2.6 \(\times\) 10\({}^{-101}\) and log2 FoldChange \(-\)2.05, FDR = 1.7 \(\times\) 10\({}^{-168}\), respectively) (Figure S2a and Table S3). These results were further validated by real-time qPCR of CD44 KO Clones #28 and #3 (Figure 4a).