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. 2014 Oct;12(10):1416-29.
doi: 10.1158/1541-7786.MCR-13-0629. Epub 2014 Jun 24.

CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility

Yushan Kim  1 , Sanjay Kumar  2
Affiliations

CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility

Yushan Kim et al. Mol Cancer Res. 2014 Oct.

Abstract

The high-molecular-weight glycosaminoglycan, hyaluronic acid (HA), makes up a significant portion of the brain extracellular matrix. Glioblastoma multiforme (GBM), a highly invasive brain tumor, is associated with aberrant HA secretion, tissue stiffening, and overexpression of the HA receptor CD44. Here, transcriptomic analysis, engineered materials, and measurements of adhesion, migration, and invasion were used to investigate how HA/CD44 ligation contributes to the mechanosensing and invasive motility of GBM tumor cells, both intrinsically and in the context of Arg-Gly-Asp (RGD) peptide/integrin adhesion. Analysis of transcriptomic data from The Cancer Genome Atlas reveals upregulation of transcripts associated with HA/CD44 adhesion. CD44 suppression in culture reduces cell adhesion to HA on short time scales (0.5-hour postincubation) even if RGD is present, whereas maximal adhesion on longer time scales (3 hours) requires both CD44 and integrins. Moreover, time-lapse imaging demonstrates that cell adhesive structures formed during migration on bare HA matrices are more short lived than cellular protrusions formed on surfaces containing RGD. Interestingly, adhesion and migration speed were dependent on HA hydrogel stiffness, implying that CD44-based signaling is intrinsically mechanosensitive. Finally, CD44 expression paired with an HA-rich microenvironment maximized three-dimensional invasion, whereas CD44 suppression or abundant integrin-based adhesion limited it. These findings demonstrate that CD44 transduces HA-based stiffness cues, temporally precedes integrin-based adhesion maturation, and facilitates invasion.

Implications: This study reveals that the CD44 receptor, which is commonly overexpressed in GBM tumors, is critical for cell adhesion, invasion, and mechanosensing of an HA-based matrix.

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Conflict of interest statement

The authors do not disclose any conflicts of interest.

Figures

Figure 1
Figure 1. CD44 is frequently overexpressed in glioblastoma multiforme tumors, and correlated with misregulation of other proteins involved in CD44-mediated pathways
(A) Transcriptomic analysis of The Cancer Genome Atlas (TCGA) microarray data: HA synthases (HAS); hyaluronidases (HYAL); the lecticans aggrecan (ACAN), brevican (BCAN), neurocan (NCAN), and versican (VCAN); the tenascins tenascin-C (TNC), tenascin-N (TNN), and tenascin-R (TNR); CD44; RHAMM (HMMR); the ERM proteins (EZR, RDX, MSN), and the ankyrins (ANK). Percentiles are represented by boxes (25, 50, 75), whiskers (10, 90), and dashes (1, 99). Bottom panel: analysis of gene expression correlation with CD44. Red-blue heat map indicates value of Pearson product-moment correlation coefficient between CD44 expression and each gene, and p values indicate the statistical significance of the correlation. (B) Expression of proteins relevant to hyaluronic acid (HA)-based brain ECM and associated downstream signaling. HA synthase proteins synthesize HA, while hyaluronidase enzymes degrade HA. Other ECM components that interact with HA include lecticans, which bind to both tenascins and HA to form a mesh-like matrix. CD44 and RHAMM are the main HA receptors. Intracellularly, CD44 links to the actin cytoskeleton through the ERM (ezrin-radixin-moesin) family proteins, and ankyrins. (C) Analysis of expression of genes encoding other extracellular matrix components previously determined to be key for glioblastoma multiforme progression: fibronectin (FN1), laminin α-5 (LAMA5), laminin α-3 (LAMA3), and vitronectin (VTN).
Figure 2
Figure 2. U373-MG cell adhesion to hyaluronic acid (HA) hydrogels is CD44- and stiffness-dependent
(A) Adhesion of cells on 4.6 kPa hydrogels at short (0.5 h) time scales. Control cells or CD44 knockdown (k.d.) cells were allowed to attach to substrates in the absence or presence of adhesion blockers, including α-CD44 neutralizing antibody, IgG isotype control antibody, or soluble RGD peptide; then centrifuged to induce detachment of weakly adhered cells. (B) Adhesion of cells on 4.6 kPa hydrogels at longer (3 h) time scales. (C) Adhesion of cells on bare HA hydrogels as a function of stiffness after 0.5 h adhesion time. (D) Adhesion of cells on RGD-functionalized HA hydrogels as a function of stiffness after 0.5 h adhesion time. #p<0.05 differences between stiffnesses by Tukey-Kramer, *p<0.05 by Student’s t-test.
Figure 3
Figure 3. U373-MG glioma cell spreading and morphology on HA-based hydrogel is stiffness-dependent
(A–B) Cell spread area after 24 h of adhesion on bare HA hydrogels (A), or HA hydrogels functionalized with RGD (B). Hydrogel shear modulus is displayed on the x-axis. A,B,C statistical families show p<0.05 from Dunn’s test for multiple comparison of non-normally distributed data. Boxes represent 25th and 75th percentiles, whiskers represent 10th and 90th percentiles. (C) Cell morphology of U373-MG human glioblastoma cells on HA hydrogel in the absence of integrin-based binding differs from cells spread on HA functionalized with RGD. Colors in top row represent localization of CD44 (red), F-actin (phalloidin, green) and nuclear DNA (DAPI, blue). Bottom row depicts localization of vinculin. Scale bar represents 50 μm. (D) Cell morphology of U87-MG human glioblastoma cells with same conditions described in (C).
Figure 4
Figure 4. U373-MG glioma cell motility on HA-based hydrogel is CD44- and stiffness-dependent
(A) Cell migration speed 12–18 h after seeding on bare HA hydrogels and on RGD-functionalized HA hydrogels. Hydrogel shear modulus is displayed on the x-axis. A,B,C statistical families show p<0.05 Dunn’s test for multiple comparison of non-normally distributed data. Boxes represent 25th and 75th percentiles, whiskers represent 10th and 90th percentiles. For pairwise comparisons between cell speeds on HA and HA-RGD hydrogels at a given shear modulus, *p<0.1, **p<0.05 by Student’s t-test. (B) Comparison of cell migration speed of control U373-MG cells with CD44 knockdown counterparts on HA hydrogels of varying RGD concetration. Comparison could not be made on bare HA hydrogels due to insufficient adhesion of CD44 knockdown cells in the absence of RGD functionalization. With HA functionalization of 1 μM RGD, very few CD44 knockdown cells attached. At all other RGD concentrations tested, no statistical difference in the migration speed between the two cell lines was found. Under all conditions tested, differences between control and CD44 knockdown cell speeds were not statistically significant. (C) Persistence parameter of random cell motility paths on bare HA and RGD-functionalized hydrogels. *p<0.1, **p<0.05 by Student’s t-test. (D) Still images taken from time lapse microscopy. Each column represents a 0.5 h interval. On bare HA of 0.95 kPa, cells consistently remain rounded. On bare HA of 6.9 kPa, cells extend small protrusions that rupture quickly, such as the one indicated by white arrows, which lead to productive migration. In contrast, cells on stiff RGD+HA hydrogels exhibit broad, stable lamellipodia. Scale bar represents 25 μm.
Figure 5
Figure 5. U373-MG glioma cell invasion through transwell membranes is promoted by HA-CD44 adhesion, but not by fibronectin-integrin adhesion
(A) Incubating control cells plated on HA+BSA coated transwells with CD44-neutralizing antibody drastically reduces invasion, demonstrating that interaction with HA is CD44-dependent. (B) Incubating CD44 knockdown cells with soluble RGD peptide on membranes coated with 3.7 μg/mL fibronectin strongly attenuates invasion, demonstrating that adhesion to fibronectin is integrin-dependent. #p<0.05 multiple comparison by Tukey-Kramer, *p<0.05 by Student’s t-test. (C) Percent of control or CD44 knockdown U373-MG glioma cells that invaded through 8 μm pores in membranes coated with combinations of HA, varying concentrations of fibronectin (FN) and BSA, or with HA dissolved in the cell media (soluble HA). Statistically significant differences of p<0.05 from multiple comparisons within cell line groups by Tukey-Kramer are grouped by A,B,C statistical families. *p<0.05 t-test pairwise comparisons of cell lines on the same substrate. (D) Representative phase contrast images of cells either above or below clear transwell membranes (top row), and GFP-expressing cells below Fluoroblok membranes (bottom row). Both types of membranes have 8 μm pores, and images were taken 2.5 h after cell seeding. Scale bars represent 100 μm.
Figure 6
Figure 6. Proposed model for influence of adhesion strength on cell invasion
We propose a model for cell invasion that involves a balance between formation and turnover of cell adhesions. (A) With little to no cell adhesion to the substrate, extrusion of the cell body through a confined space is not possible. (B) With an optimal balance between adhesion and detachment, as is the case with the relatively labile CD44-HA interaction, cells have the highest invasion potential. (C) When abundant integrin-based adhesion is present, the cells spread extensively and adhere robustly on the substrate but do not invade.
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