The Relationship of Hyaluronic Acid-CD44 on Cancer Progression and Hyaluronic Acid-Based Self-Assembled Nanoparticle Roles of Hyaluronic Acid-CD44 on Cancer Progression and Hyaluronic Acid-Based Self-Assembled

Cancer cells have the ability to multiply uncontrollably and metastasize. For metastasizing, they need to change the extracellular matrix (ECM) structure. One of the most important main components of the extracellular matrix is hyaluronic acid (HA). HA fragments and the HA receptor CD44 are very high in cancer cells. The HA-CD44 receptor relationship in cancer cells is one of the mechanisms that are used to target nanoparticles. Self-assembled nanoparticles are self-organizing nanoparticles. Anticancer drugs that have low solubility in water are transformed with self-assembled nanoparticles and provided with a hydrophilic structure and targeted towards the CD44 receptors that are highly expressed in cancer cells. Anti-cancer drugs of self-assembled HA nanoparticle form can stay in circulation without being destroyed for a long time with surfactant or polymer structures. Additionally, self-assembled HA nanoparticles are more effective on cancer cells and less cytotoxicity on healthy cells. The lifespan quality of cancer patients may be increased by usage of nanoparticles such as target-oriented self-assembled HA nanoparticles.

its status as a main structural component, hyaluronic acid is also effective on several cellular functions. It plays a role in important cellular activities such as cell differentiation, growth, cell adhesion, cell migration and proliferation [6].
It is known that low molecular weight HA (LMW-HA) accumulates in various types of cancer. LMW-HA has been associated with various cellular processes such as proliferation, invasion and angiogenesis [9]. Progressive weight reduction of HA fragments can decrease ECM density and cancer cell invasion get easier to other tissues. In addition to this, inflammation, angiogenesis, metastasis and multidrug resistance (MDR) increase [10]. While LMW-HA contributes to favoring cancer cells, HMW-HA has the opposite effect [11], just as in its protective effect against cancer in naked mole rats.
Naked mole rats have a much longer lifespan in comparison to other rodents. While the average lifespan of mice and other rodents is about 4 years, this time is 32 years for naked mole rats. Several scientists reported that HA, provides animals resistance to cancer [12]. The reason for these rats to not get cancer is the HMW-HA, they possess which is approximately 5 times larger than that in mice and humans. This special HMW-HA is synthesized by fibroblasts forms a tight density around the cells. It is believed that this cloud of HMW-HA that surrounds cells prevents not only transformation of normal cells into cancer cells but also metastasis. The thing which makes this hyaluronic acid special is the structural differentiation of HAS2 synthase, which is one of the enzymes that synthesize hyaluronic acid, in difference to the case in humans [13,14]. Such a large HMW-HA cannot be synthesized in humans [9].
In addition to the presence of HMW-HA, prevention of cancer formation in naked mole rats also involves the mechanisms of p16 induction (HMW-HA mediated) and early contact inhibition [14][15][16]. HA is different to other GAGs. HA is not synthesized from the Golgi apparatus as a single protein such as proteoglycan. It is synthesized by mediation of the HA synthases that are found in the inner surface of the plasma membrane (HAS1, HAS2, HAS3).
While HAS1 and HAS2 synthesize HA with high molecular weights, HAS3 is responsible for synthesis of HA with low molecular weights Receptor (LYVE) and layilin and some ECM molecules such as collagen VI and Proteoglycans [4,11,17]. As the receptor CD44 has appeared to be associated with cancer more in comparison to other receptors, it has been researched in more detail.
CD44 is found in endothelial cells, leukocytes, epithelial cells, fibroblasts, keratinocytes, hematopoietic cells and various cancer cells [8,18]. It has a glycoprotein structure, and it passes the membrane along its axis. It has 7 extracellular domains, a transmembrane domain and a cytoplasmic domain. While the N-terminal is found in the extracellular region, the C-terminal is inside the cell.
There is a "link module" motif in the N-terminal where HA and HA-bonded proteoglycans can bond [8]. The end of the C-terminal is associated with cytoskeleton proteins [2]. CD44 has great significance for cell adhesion during the cell migration and tissue formation [2]. The adhesion and spreading activities of lymphocytes are also associated with CD44 [19]. It plays a role in extracellular vesicle interaction [20]. Besides these, it is known to play a role in physiological processes such as hematopoiesis and extremity development [20].
CD44 has several isoforms that are formed by alternative splicing of one gene in the 20-exon region. This diversity increases by post-translational modifications (N and O-glycosylation) [2].
CD44 receptors that are formed by alternative splicing taking place in the 6-15 exons of the CD44 gene are known as "variants" (CD44v) [21]. The standard CD44 isoform is CD44s [2]. CD44v is expressed in extraordinary circumstances such as development and tumors. Among these, high expression of CD44v6 in different types of cancer has been reported, and it is known that it is effective in cancer formation and progression [8,20]. Hyaluronic acid is not the only ligand of the CD44 receptor, and CD44 gains the ability to bond with different ligands by exposure to post-translational modifications. These include collagen, fibronectin, laminin, fibrin, osteopontin (OPN) and serglycin [11]. It was shown that HA, HA synthase, Hyaluronidases and hyaluronic acid receptors increase in various types of cancer [22].
However, their mechanisms in the cancer formation process have not been enlightened yet [5,22]. Nevertheless, some possible mechanisms related to this issue have been proposed. The first one of these was associated with the Warburg effect that is seen in cancer cells [11]. Accordingly, cancer cells prefer the way of anaerobic respiration to be able to produce ATP and require too much glucose to be able to produce sufficient energy. This hyperglycemic envi- CD44v6 shows an anti-apoptotic effect as a result of its collaboration with receptor tyrosine kinases, contributes to drug resistance and affects HA synthesis in an increasing direction [11].
The HA-CD44 ligand/receptor relationship is utilized nowadays to design nanoparticles that are used for cancer treatment. Anticancer drug are used together with carrier nanoparticles, the time they spend in circulation increases, and they directly target cancer cells. This is why they become more effective in lower doses in comparison to the dose used in chemotherapy, and they affect healthy cells to a minimal extent, some side effects that are seen in chemotherapy patients are eliminated/reduced. This increases the quality of life of cancer patients [2,23]. There are various HA-based nanomaterials that are used for the purpose of cancer treatment.
These include polymeric drug-conjugated HA, nanomaterials (such as: micelles, polymersome, hydrogels)and inorganic nanoparticle materials [24]. One example of these may be self-assemble nanoparticles. Self-assembled HA nanoparticles are drug carriers that are frequently utilized in cancer treatment, because the expression of the HA receptor CD44 in cancer cells is very high [25].
Self-assembled nanoparticles consist of three parts. These are the surface, shell and core [26]. Such nanoparticles have a hydrophobic core and a hydrophilic shell. The hydrophobic core serves collection of drugs with a hydrophobic structure. The outer surface of the hydrophilic shell has surfactants, and these constitute the surface part of the nanoparticle [23,26]. These molecules found in the nanoparticle surface are monofunctional long-chain molecules such as amines, phosphines, carboxylates and thiols.
These molecules provide nanoparticle stability. Among these, the most frequently preferred one is Polyethylene glycol (PEG) [26]. As nanoparticles with a PEG structure are not noticed by phagocytic cells, they have a lower rate of opsonization, and they show a more effective accumulation in cancerous tissues [27].
Self-assembled nanoparticles show a self-forming behavior in aqueous environments due to their amphiphilic structures.
Anticancer drugs that have low solubility in water are transformed with self-assembled nanoparticles and provided with a hydrophilic structure. Various polymers and surfactants are used in the surface part of nanoparticles [26]. These provide the nanoparticle with a surface charge. In addition to this, they passively play a role in carrying the nanoparticle to the targeted cancer cells [28] and allow it to stay in circulation [26]. Additionally, structures such as antibodies, receptors, small organic molecules that are added to the surface parts of nanoparticles play a role in recognition of nanoparticles by tumor tissues, as well as their bonding and intake into the cell [27,29]. One of such molecules is HA, which is used to target nanoparticles towards cancer cells that are known to show high expression of CD44 [30,31]. The surfaces of cancer cells are highly rich in terms of CD44 receptors. Self-assembled HA nanoparticles that effectively access the tumor tissue are bonded with the CD44 receptors that are found in abundance on the surfaces of cancer cells.
The Hyal-2 that is found on the cell surface starts degradation of the nanoparticle structure by degrading the HA molecules in this structure. Self-assembled-HA nanoparticles are taken into the cancer cells by CD44 receptor-mediated endocytosis.The partly degraded nanoparticles are degraded even more by the Hyal-1 inside the cell, and the anticancer agent is dispersed within the cell [32]. There are various cancer studies that have been carried out on self-assembled HA nanoparticles. The study by Choi et al. examined the properties of self-assembled HA nanoparticles and revealed that HA nanoparticles have a high ability of being targeted towards cancer cells [25]. Choi et al. introduced self-assembled HA nanoparticles with an without PEG and it was reported that PEG-coated self-assembled HA nanoparticles stayed in circulation for a longer time, and they showed higher accumulation in the tumor tissue in comparison to the HA nanoparticles without PEG coating [33].
In a similar study, PEG was added to the structure of selfassembled HA nanoparticles to be used in carrying the anticancer agents, doxorubicin and camptothecin, to cancer tissues. It was shown that the nanoparticle forms were more effective in comparison to those that were freely provided [32]. In the study by Huang et al. (2013), docetaxel-PLGA-HA nanoparticles showed an anti-tumor effect by reducing systemic toxicity in breast cancer mice [23]. Self-assembled HA nanoparticles that were used in another study were proven to block CD44 receptors in colon cancer cells [31]. In the study by Ganesh et al. [34], siRNA trapped in selfassembled nanoparticles inhibited the high expression of CD44 [34].
Self-assembled HA nanoparticles may be easily targeted towards cancer cells with high CD44 expression. Due to the surfactants or polymers added to the nanoparticle surfaces such as PEG, they can stay in circulation for a long time without opsonization by the reticuloendothelial system, and they show a more effective cytotoxic influence on cancer cells. The lifespan and quality of life of cancer patients may be increased by usage of nanoparticles such as target-oriented self-assembled HA nanoparticles.