The cell-surface glycoprotein CD44 is involved in a multitude of important physiological functions including cell proliferation, adhesion, migration, hematopoiesis, and lymphocyte activation. The diverse physiological activity of CD44 is manifested in the pathology of a number of diseases including cancer, arthritis, bacterial and viral infections, interstitial lung disease, vascular disease and wound healing. This diversity in biological activity is conferred by both a variety of distinct CD44 isoforms generated through complex alternative splicing, posttranslational modifications (e.g., N- and O-glycosylation), interactions with a number of different ligands and the abundance and spatial distribution of CD44 on the cell surface. The extracellular matrix glycosaminoglycan hyaluronan (HA) is the principle ligand of CD44. This review focuses both CD44-hyaluronan dependent and independent CD44 signaling and the role of CD44-HA interaction in various pathophysiologies. The review also discusses recent advances in novel treatment strategies that exploit the CD44-HA interaction either for direct targeting or for drug delivery.

Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3–8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority (∼75–80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3–8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0–4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a ∼60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP.

4-Methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility, and invasion of DU145, PC3-ML, LNCaP, C4-2B, and/or LAPC-4 cells. At IC₅₀ for HA synthesis (0.4 mmol/L), 4-MU induced >3-fold apoptosis in prostate cancer cells, which could be prevented by the addition of HA. 4-MU induced caspase-8, caspase-9, and caspase-3 activation, PARP cleavage, upregulation of Fas-L, Fas, FADD and DR4, and downregulation of bcl-2, phosphorylated bad, bcl-XL, phosphorylated Akt, phosphorylated IKB, phosphorylated ErbB2, and phosphorylated epidermal growth factor receptor. At IC₅₀, 4-MU also caused >90% inhibition of NF-κB reporter activity, which was prevented partially by the addition of HA. With the exception of caveolin-1, HA reversed the 4-MU–induced downregulation of HA receptors (CD44 and RHAMM), matrix-degrading enzymes (MMP-2 and MMP-9), interleukin-8, and chemokine receptors (CXCR1, CXCR4, and CXCR7) at the protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU–induced apoptosis and inhibition of cell growth and interleukin-8, RHAMM, HAS2, CD44, and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (>3-fold) when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry, or body weight. Tumors from 4-MU–treated animals showed reduced microvessel density (∼3-fold) and HA expression but increased terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive cells and expression of apoptosis-related molecules. Therefore, the anticancer effects of 4-MU, an orally bioavailable and relatively nontoxic agent, are primarily mediated by inhibition of HA signaling. Cancer Res; 70(7); 2613–23

Hyaluronic acid (HA), a nonsulfated glycosaminoglycan, regulates cell adhesion and migration. Small HA fragments (3-25 disaccharide units) induce neovascularization. We investigated the effect of HA and a HA fragment (10-15 disaccharide units, F1) on primary human endothelial cells (ECs). Human pulmonary ECs (HPAEC) and lung microvessel ECs (HMVEC-L) bound HA (K(d) approximately 1 and 2.3 nm, respectively) and expressed 17,780 and 16,690 HA binding sites, respectively. Both ECs showed HA-mediated cell adhesion; however, HMVEC-L was 1.5-fold better. Human umbilical vein ECs neither bound HA nor showed HA-mediated adhesion. All three ECs expressed CD44 ( approximately 110 kDa). The expression of receptor for HA-mediated motility (RHAMM) (approximately 80 kDa) was the highest in HMVEC-L, followed by HPAEC and human umbilical vein ECs. RHAMM, not CD44, bound HA in all three ECs. F1 was better than HA and stimulated a 2. 5- and 1.8-fold mitogenic response in HMVEC-L and HPAEC, respectively. Both HA and F1 induced tyrosine phosphorylation of p125(FAK), paxillin, and p42/44 ERK in HMVEC-L and HPAEC, which was blocked by an anti-RHAMM antibody. These results demonstrate that RHAMM is the functional HA receptor in primary human ECs. Heterogeneity exists among primary human ECs of different vascular origins, with respect to functional HA receptor expression and function.

The tumor cell–derived hyaluronidase (HAase) HYAL-1 degrades hyaluronic acid (HA) into proangiogenic fragments that support tumor progression. Although HYAL-1 is a critical determinant of tumor progression and a marker for cancer diagnosis and metastasis prediction, it has not been evaluated as a target for cancer therapy. Similarly, sulfated hyaluronic acid (sHA) has not been evaluated for biological activity, although it is an HAase inhibitor. In this study, we show that sHA is a potent inhibitor of prostate cancer. sHA blocked the proliferation, motility, and invasion of LNCaP, LNCaP-AI, DU145, and LAPC-4 prostate cancer cells, and induced caspase-8–dependent apoptosis associated with downregulation of Bcl-2 and phospho-Bad. sHA inhibited Akt signaling including androgen receptor (AR) phosphorylation, AR activity, nuclear factor κB (NFκB) activation, and VEGF expression. These effects were traced to a blockade in complex formation between phosphoinositide 3-kinase (PI3K) and HA receptors and to a transcriptional downregulation of HA receptors, CD44, and RHAMM, along with PI3K inhibition. Angiogenic HA fragments or overexpression of myristoylated Akt or HA receptors blunted these effects of sHA, implicating a feedback loop between HA receptors and PI3K/Akt signaling in the mechanism of action. In an animal model, sHA strongly inhibited LNCaP-AI prostate tumor growth without causing weight loss or apparent serum-organ toxicity. Inhibition of tumor growth was accompanied by a significant decrease in tumor angiogenesis and an increase in apoptosis index. Taken together, our findings offer mechanistic insights into the tumor-associated HA–HAase system and a preclinical proof-of-concept of the safety and efficacy of sHA to control prostate cancer growth and progression. Cancer Res; 71(12); 4085–95. ©2011 AACR.