Interactions between Hyaluronan and Its Receptors (CD44, RHAMM) Regulate the Activities of Inflammation and Cancer

Abstract

The glycosaminoglycan hyaluronan (HA), a major component of extracellular matrices, and cell surface receptors of HA have been proposed to have pivotal roles in cell proliferation, migration, and invasion, which are necessary for inflammation and cancer progression. CD44 and receptor for HA-mediated motility (RHAMM) are the two main HA-receptors whose biological functions in human and murine inflammations and tumor cells have been investigated comprehensively. HA was initially considered to be only an inert component of connective tissues, but is now known as a "dynamic" molecule with a constant turnover in many tissues through rapid metabolism that involves HA molecules of various sizes: high molecular weight HA (HMW HA), low molecular weight HA, and oligosaccharides. The intracellular signaling pathways initiated by HA interactions with CD44 and RHAMM that lead to inflammatory and tumorigenic responses are complex. Interestingly, these molecules have dual functions in inflammations and tumorigenesis. For example, the presence of CD44 is involved in initiation of arthritis, while the absence of CD44 by genetic deletion in an arthritis mouse model increases rather than decreases disease severity. Similar dual functions of CD44 exist in initiation and progression of cancer. RHAMM overexpression is most commonly linked to cancer progression, whereas loss of RHAMM is associated with malignant peripheral nerve sheath tumor growth. HA may similarly perform dual functions. An abundance of HMW HA can promote malignant cell proliferation and development of cancer, whereas antagonists to HA-CD44 signaling inhibit tumor cell growth in vitro and in vivo by interfering with HMW HA-CD44 interaction. This review describes the roles of HA interactions with CD44 and RHAMM in inflammatory responses and tumor development/progression, and how therapeutic strategies that block these key inflammatory/tumorigenic processes may be developed in rodent and human diseases.

Keywords: CD44; RHAMM; cancer; hyaluronan; inflammation.

Figure 1

Native polymeric HA and degraded HA fragments. High molecular weight (HMW ≥ 500 disaccharide units; [β-1, 4-GlcUA-β-1, 3-GlcNAc]n synthesized in the normal homeostatic condition is degraded by free radicals and hyaluronidases (HYAL1-2) during inflammation/tumorigenesis when tissue injury occurs. These fragments are ≤50 disaccharide units. As a result, the fragments of different molecular weights have different biological functions. For example, intermediate fragments (30–500 kDa) can stimulate cell proliferation while smaller fragments <50 kDa promote cell migration. HA oligosaccharides down to three disaccharides can still bind to CD44.

Figure 2

(A) Mouse CD44 and RHAMM gene/protein structures in mice. Model structure of alternative splicing in CD44. CD44 pre-mRNA is encoded by 20 exons in mouse and 19 exons in human being. The common standard CD44s (hematopoietic) form contains no extra exons, and the protein has a serine motif encoded in exon 5 that can initiate synthesis of a chondroitin sulfate or dermatan sulfate chain. Alternative splicing of CD44 predominantly involves variable insertion of 10 extra exons with combinations of exons 6–15 and spliced in v1–v10 into the stem region, of which v3 encodes a substitution site for a heparan sulfate chain. Variable numbers of the v exons can be spliced in epithelial cells, endothelial cells, and inflammatory monocytes and also are upregulated commonly on neoplastic transformation depending on the tissue. (B) Model structure of alternatively spliced CD44 proteins. The CD44 protein is composed of an extracellular N-terminal domain, a stem region in the extracellular domain close to the transmembrane region, where the variant exon products (red/violet circles) are inserted, the transmembrane region, and the carboxyl terminal cytoplasmic tail. There are multiple sites for N-glycosylation (purple circles) and O-glycosylation (orange circles), and a sulfation domain. The N-terminal portion contains highly conserved disulfide bonds as well as 2 BX7B motifs, both of which are essential for HA binding. CD44 is subjected to extensive glycosylation, sulfation, and attachment of GAGs that contribute to regulation of the HA-binding activity. The C-terminal cytoplasmic tail contains several phosphorylation sites that regulate the interaction of CD44 with the cytoskeletal linker proteins, as well as with SRC kinases. (C) RHAMM exon structure. The full-length protein (85 kDa in human beings) is largely associated with microtubule formation during the cell cycle progression. Three isoforms are generated by alternative splicing of exon 4, 5, or 13. Loss of exon 4 disrupts association with microtubules and results in the appearance of RHAMM in the cell nucleus. N-terminal truncations that may be generated by a posttranslational mechanism are constitutively present in some aggressive breast cancer cell lines and tumors. These accumulate in the nucleus and on the cell surface. (D) The secondary structure of RHAMM. RHAMM can self-associate to form random coiled coils (132).

Figure 3

The involvement of HA and CD44 in cell survival and differentiation. (A) Model for the involvement of CD44v6 and Met due to autocrine TGF β1 signaling in lung fibrogenic fibroblasts. The repetitive lung injury in pulmonary fibrosis results in overexpression of TGFβ1 and TGFβ1-induced autocrine signaling that induces a sustained expression of CD44v6 and its co-receptor c-Met. This activates fibrogenic lung fibroblasts with subsequent increased collagen matrix synthesis. Therefore, TGF β1-induced CD44v6 and Met can have a crucial role for the sustained fibrogenic activation of lung fibroblasts. The CD44-phosphorylated ERM complex initiates activation of transforming growth factor-β receptor 1 and 2 (TGFβRI and II) and the downstream SMAD signaling complex, which contribute to fibrosis. (B) Model for involvement of periostin in HA-CD44-mediated cell survival and differentiation. Matricellular protein [periostin (PN)] binding to β1 or β3-integrin activates FAK, which activates downstream MAPK/Erk and PI3K/Akt to regulate cardiac valve cell growth, survival, differentiation into fibroblasts, and matrix organization (maturation). PN binding to β3-integrin also activates Has2 mRNA expression, Has2 phosphorylation, and HA synthesis. The interaction of HA with CD44, may, in turn, amplify the downstream effects of PN on heart valve cushion cell differentiation/maturation processes. (C) Cleavage of the extracellular domain is accompanied by the cleavage of the intracellular domain (ICD) by the presenilin-γ-secretase complex. The CD44 ICD acts together with CBP or p300 as a transcription factor and promotes CD44 transcription and extracellular matrix production.

Figure 4

CD44- and CD44v-induced RTK for apoptosis resistance. (A) CD44-HA binding, accompanied by activation of CD44-associated SRC, ezrin phosphorylation, and PI3K activation leads to the lipid-raft-integrated assembly of a complex that includes heat shock protein 70 (HSP70), the co-chaperone CDC37, Rho-GEF, Grb2/VAV2, and Gab-1/PI3-kinase (PI3K), which promotes phosphorylation and activation of the receptor tyrosine kinases (RTKs), including ERBB2, ErbB3, EGFR, IGF1R-β, PDGFR-β, and c-MET. CD44-HA binding initiates cross-talk between RTKs, non-RTKs [SRC (Src)] and linker proteins. Studies have indicated that ErbB2 most likely complexes with CD44 via interactions with Grb2 and Vav2, whereas the interaction of PI3K and CD44 is mediated by Gab-1. PI3K activates Akt and downstream anti-apoptotic events, which contribute to drug resistance. However, HA and PI3K stimulate MDR1 expression, and the stimulatory effects of PI3K would be mainly due to its feedback stimulation of HA production by a positive feedback loop. Studies indicate that MDR1 is associated with CD44 in lipid microdomain and can be linked via CD44 with the actin cytoskeleton so that expression of both CD44 and MDR1 are concomitantly regulated. (B) Particularly, in colorectal cancer, the CD44–ERBB2 complex provides a strong apoptotic resistance through stimulation of cyclooxygenase 2 (COX2) transcription via PI3K and β-catenin. COX2-induced PGE2 stimulates HA synthesis and HA-CD44 signaling. CD44v6 also binds hepatocyte growth factor (HGF) and presents it to c-MET. Activation of MET and the downstream signaling cascades require sustained activation of CD44-associated phosphorylated ezrin, radixin, and moesin (ERM) and SRC (Src) signaling via the Ras-MAPK and the PI3K-Akt pathway. Ras-Erk pathway can augment CD44v6 synthesis through a feedback loop between CD44v6 and c-Met/Ras-Erk pathway.

Figure 5

Cross-talk between CD44 and RHAMM interaction with HA affects physiological and cellular functions. Green Track is for extracellular RHAMM signaling involving CD44-HA-mediated pathways. Red track is for intracellular RHAMM signaling. Studies suggest a model for functions of CD44 and RHAMM. The interaction of HA-CD44-RHAMM affects physiological and cellular functions. Cell surface RHAMM interacts with CD44, HA, and growth factor receptors (GFR) to activate protein tyrosine kinase signaling cascades that activate the ERK1/2 MAP kinase cascade in a c-Src/Raf-1/MEK-1/ERK1/2 dependent manner (depicted in green track). In the absence of intracellular RHAMM, this signaling can stimulate the transcription of motogenic effectors to regulate a mitogenic response (cell proliferation/random motility). In the presence of intracellular RHAMM (red track), MEK-1/p-Erk1/2 also binds to a number of protein partners that allows activated RHAMM to enter the nucleus to regulate functions of microtubule dynamics via centrosome structure/function, and cell cycle progression via AURKA, a targeting protein for XkIp2 (TPX2). Activated RHAMM also controls expression of genes involved in cell motility such as PAI-1 and MMP-9.

Figure 6

Drug targeting approaches to exploit HA-CD44 interaction (C) in cancer treatment. (A) HMWHA-CD44v interaction induces inflammation and tumor growth; most common blocking reagents against CD44 isoforms: (B) LMW HA inhibits the binding of HMWHA; (C) sol-CD44 competes with HMWHA to bind with CD44v; (D). CD44-blocking monoclonal antibody (CD44-mAb); (E) antibodies against CD44v6; (F) peptides blocking binding of HA with CD44v6; (G) enzymatic hydrolysis by hyaluronidase cleaves the HMWHA to small fragments and blocks the HA-CD44v6 interaction; (H) targeting HA receptor with HA-drug conjugate; (I) targeting “HA receptor” with “HA nanoparticles.”

Figure 7

Schematic illustration of cellular uptake of plasmid DNA/Tf-PEG-PEI (nanoparticles) polyplexes, their shielding from non-specific interaction, and the mechanism of action of shRNA. Internalization of PEG-shielded and [transferrin receptor (Tf-R)]-targeted polyplexes into target cells occurs by receptor-mediated endocytosis after association of polyplex ligand Tf to Tf-R present on the target cell plasma membrane. Internalized particles are trafficked to endosomes followed by endosomal release of the particles and/or the nucleic acid into cytoplasm. Released siRNA will form a RNA-induced silencing complex and will be guided for cleavage of complementary target mRNA in the cytoplasm. SiRNA (antisense) guide strand will direct the targeted RNAs to be cleaved by RNA endonuclease. Finally, plasmid/Tf-PEG-PEI-nanoparticles delivery in the target cell shows reporter gene expression and activity. The normal tissue cells are not affected because they do not make the targeted CD44 variant. Tf-PEG-PEI nanoparticle coated plasmids (pSico-CD44v6 shRNA/pFabpl-Cre) circulating in blood accumulate at tumor regions enhanced by the EPR effect. Endocytosis mediated by ligand-receptor interactions occurs because the nanoparticles are coated with the Tf-ligand for the Tf-R receptor on the tumor cell surface.

Figure 8

Exploitation of HA-CD44 interaction for anticancer therapy. Left panel shows a schematic diagram for cancer cell internalization of HA conjugated to a drug inhibitor of DNA synthesis. CD44 on the membrane traps the HA-drug conjugate and efficiently internalizes it by endocytosis and forms an endosomal vesicle. This is then transported to the lysosome and fused, and the internalized HA is degraded by hyaluronidase 1 (Hyal-1) into small HA oligosaccharides and then to monosaccharides by lysosomal glycosidases, which ultimately releases the conjugated drug. The drug then goes to the nucleus and inhibits the DNA synthesis. Right panel shows targeting the CD44v6mRNA in cancer cells by CD44v6 shRNA. Plasmids that produce CD44v6 shRNA are coated with transferrin present on the outer surface of the nanoparticles. The transferrin molecules then target the transferrin receptors present in high amounts on the cancer cells. Upon internalization, an endosome forms and the plasmids are released to the nucleus where CD44v6 shRNA is produced by DNA pol III. The shRNA is then exported out of the nucleus by exportin, and the dicer enzyme converts shRNA into CD44v6 siRNA. One of the strands of siRNA will bind to CD44v6 mRNA and form a RISC (RNA-induced silencing complex) that is ultimately degraded. Adapted from our article in the International Journal of Cell Biology.