Dissecting the role of hyaluronan synthases in the tumor microenvironment

Abstract

The tumor microenvironment is becoming a crucial factor in determining the aggressiveness of neoplastic cells. The glycosaminoglycan hyaluronan is one of the principal constituents of both the tumor stroma and the cancer cell surfaces, and its accumulation can dramatically influence patient survival. Hyaluronan functions are dictated by its ability to interact with several signaling receptors that often activate pro-angiogenic and pro-tumorigenic intracellular pathways. Although hyaluronan is a linear, non-sulfated polysaccharide, and thus lacks the ability of the other sulfated glycosaminoglycans to bind and modulate growth factors, it compensates for this by the ability to form hyaluronan fragments characterised by a remarkable variability in length. Here, we will focus on the role of both high and low molecular-weight hyaluronan in controlling the hallmarks of cancer cells, including cell proliferation, migration, metabolism, inflammation, and angiogenesis. We will critically assess the multi-layered regulation of HAS2, the most critical hyaluronan synthase, and its role in cancer growth, metabolism and therapy.

Graphical Abstract

Hyaluronan Synthase 2 (HAS2) is a key ubiquitous enzyme located at the plasma membrane that synthesizes hyaluronan and extrudes these long polysaccharides into the extracellular space. Here, we critically asses the role of HAS2 and hyaluronan in the tumor microenvironment and its regulation by a natural antisense transcript. We will also cover the function of hyaluronan in inflammation and angiogenesis.

Introduction

In every tissue, the fundamental extracellular matrix (ECM) is composed of a three-dimensional, highly dynamic structure that undergoes continuous remodeling to maintain physical support and tissue homeostasis. However, several diseases are associated with dysregulation of the subtle dynamics controlling ECM remodeling. Genetic mutations in genes involved in the synthesis of ECM components can cause severe dysfunction by decreasing the secretion of vital ECM proteins or by enhancing cellular retention or degradation [1,2]. Cancer development has long been depicted as a progression of serial genetic mutations in a tumor cell mass. However, the biology of tumors is much more complex, and it is highly influenced by their tumor microenvironment, which has emerged as a critical player in regulating tumorigenesis. The extracellular matrix (ECM) includes hyaluronan [3,4] and several functional proteins that regulate cellular activity, mechano-sensing [5], remodeling and regeneration [6], as well as normal physiology and disease [7,8]. The complex structure of the 3D extracellular matrix surrounding the neoplastic cells acts as an essential participant in orchestrating many aspects of tumor growth and progression including invasion, metastasis, and angiogenesis. The intricate biology of the basement membrane (BM) is also involved in evoking pathogenic mechanisms that initiate cancer and other diseases [9]. In this picture, various receptors, proteoglycans (PG), members of the inflammatory response, growth factors, and other players induce a pro- or anti-tumorigenic response. Notably, many proteoglycans exert critical roles in cancer and inflammation as they bind to various receptors and growth factors in the ECM [10,11]. The breakdown and remodeling of the ECM mediated by all these players can result in several pathologies affecting the musculoskeletal and renal systems [12–18]. Indeed, the extracellular matrix is the major driving force of nearly all human diseases and across all organs as no tissue is exempt from the “matrix” [19]. In this review, we will dissect the role of hyaluronan (HA), a ubiquitous master core component of the ECM, and the enzymes involved in its regulation with a sharp focus on their mechanism of action within the tumor microenvironment. We will elucidate the roles of HA in cancer development, its functions, and how the regulation via its three synthases (HAS1/2/3) can decide the fate of solid malignancies.

Hyaluronan: A repetitive sugar charged with biological activity

Hyaluronan is an unsulfated linear glycosaminoglycan synthesized by a wide variety of organisms, and it represents one of the major constituents of interstitial spaces and the largest polysaccharide found in vertebrates [20]. Hyaluronan is ubiquitously expressed in all cells from vertebrate species, and its expression is correlated with cell motility and tissue growth [21]. Hyaluronan consists of repeating disaccharides composed of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) connected with b-1.3- and b-1.4- glycosidic bonds. Contrary to all the other glycosaminoglycans, which are produced in the Golgi apparatus and covalently linked to protein cores, HA is synthesized in eukaryotic cells by membrane-bound synthases at the inner side of the plasma membrane which produce large HA aggregates of various molecular mass up to 3–4 MDa. Afterward, it is extruded through pore-like structures to the cell surface. Interestingly, HA is not further modified by sulfation or by epimerization of the glucuronic acid moiety to iduronic acid [20]. Hyaluronan biosynthesis is catalyzed by hyaluronan synthases (HASs) at the inner surface of the plasma membrane in eukaryotic cells (Fig. 1A). The synthases use the cytosolic substrates UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc) and extrude the growing polymer through the membrane to form elongated HA molecules (Fig. 1B) [22–24]. The reducing end of the growing chain displaces a UDP moiety when the next nucleotide sugar is added. HA, previously considered to be an inert component of the connective tissue, has instead a wide variety of physiological functions, which include the provision of a matrix for cell migration and proliferation, cell adhesion, space-filling, and joint lubrication [4,25]. Moreover, HA is necessary for the healing of a wound area as it provides a framework for the development and sprouting of blood vessels [26]. Hyaluronan exerts the majority of its biological functions by either binding to proteins present on the cell surface or secreted into the extracellular matrix. The major family of proteins that selectively binds to HA is called hyalectans [27] and is composed by four secreted proteoglycans (versican, neurocan, brevican, and aggrecan). These proteoglycans are expressed in different tissues and share homologous G1 domains that mediate the interaction with HA, and upon which, they form aggregates [28]. Versican, for example, is an active player in vascular biology and it is mostly found in soft tissues [29]. It binds HA through a mechanism highly similar to the one of aggrecan [28]. In the brain, instead, neurocan and brevican are highly expressed, and most likely they similarly anchor HA [30]. Interestingly, in a recent study, the breakdown of the ECM mediated by hyaluronidase and the consequent lower levels of HA increased glycolysis via a substantial upregulation of GLUT1 (glucose transporters) at the plasma membrane. Ultimately, this robust increase in glycolysis promotes cell motility and provides a mechanistic link between HA and glucose metabolism [31].

Fig. 1.

(A) Membrane organization of the domains of mammalian hyaluronan synthases. All the three enzymes comprise 6.5 membrane-spanning domains and long cytoplasmic loops. The cytoplasmic loops harbor two active sites, which participate in the transfer of UDP-GlcA and UDP-GlcNAc substrates. (B) Proposed organization of the various membrane-spanning domains allowing the nascent polymer to be extruded into the pericellular space. M⁺⁺ refers to metal ion cofactor. Modified from Itano and Kimata [119].

Two cell-surface HA receptors have been identified, namely, CD44, also known as homing cell-adhesion molecule and Hermes antigen, and the receptor for hyaluronan-mediated motility (RHAMM). Both receptors have pivotal roles in the biological functions of HA and are both associated with inflammation and cancer progression. CD44 is a transmembrane glycoprotein with a wide range of glycosylation splice variants. Changes in CD44 expression in many cell lines are associated with cancer initiation and progression [32]. RHAMM is mostly overexpressed in cancer and regulates cytoskeleton changes through focal adhesions thereby enhancing cell motility and transformation [33]. The interactions of HA with its receptors and binding-proteins are complicated due to the constant turnover and rapid metabolism of HA that produce hyaluronan molecules of different sizes. HA is produced at the plasma membrane as a high molecular weight (HMW-HA) molecule. After hyaluronidase cleavage and exposure to stress-induced reactive oxygen species (ROS), HA goes under depolymerization and breaks down into low molecular weight fragments (LMW-HA) and smaller oligosaccharides [34]. These various products differentially affect several cancer hallmarks like proliferation, invasion, evasion, and angiogenesis and are therefore of high interest for the development of therapeutic strategies to counteract tumor progression.

Hyaluronan roles in cancer development

In the vast majority of cancers, hyaluronan levels directly correlate with increased malignancy and poor prognosis. In prostate cancer, for example, increased HA in the tumor stroma is linked to enhanced growth and poor outcome [35]. In breast cancer, clinical studies have shown that LMW-HA expression is elevated in the cancer cells that display a higher invasive potential [36]. Moreover, the high level of stromal HA staining in HER2-positive breast tumors and peri-neoplastic stroma have been linked to lymph node-positive breast cancer and reduced survival [32]. High concentrations of LMW-HA have also been detected in the urine of high-grade bladder cancer patients [37] and in the saliva of high-stage head and neck squamous cell carcinoma patients together with elevated expression of CD44 [38]. Together, these studies suggest a strong correlation between high HA levels, particularly in the tumor stroma, and pro-tumorigenic mechanisms and neoplastic progression [39]. However, the role of HA in cancer is highly complicated and somewhat controversial as it has also been reported that some malignancies present diminished HA content. For example, poorly-differentiated squamous cell carcinomas show low HA concentration despite the constant HA expression in the tumor-associated stroma [40]. Also, stage I cutaneous melanomas show reduced levels of the HA-receptor CD44 [41].

It is essential to understand that the different hyaluronan polymers, from small fragments to large aggregates of high molecular weight, have different biological outcomes. The breakdown of HMW-HA causes the formations of smaller processed fragments that stimulate expression of pro-inflammatory cytokines and growth factors [42]. LMW-HA has well-established angiogenic and tumorigenic proprieties by altering selective signaling pathways, whereas endogenous HMW-HA has been shown to be anti-inflammatory and anti-angiogenic [43]. Decreasing LMW-HA production significantly inhibits breast cancer cell migration and invasion [36]. Moreover, excess LMW-HA in the tumor microenvironment has been shown to facilitate lymphatic metastasis via disruption of intercellular adhesion among lymphatic endothelial cells [44]. In addition, in the tumor interstitial fluid of colorectal cancer patients, LMW-HA concentrations are increased and associated with lymphatic vessel invasion by cancer cells and the formation of lymph node metastases [45]. Interestingly, LMW-HA shows more immunoreactive abilities as compared to HMW-HA.

The compelling influence that HMW-HA has on tumor biology is elegantly described in the naked mole rat. These animals show null incidence of cancer development and an extraordinary longevity compared to other rodents due to a superior concentration and size of HMW-HA (>6 MDa) in their skin [46]. Naked mole rat fibroblasts present a unique sequence of HAS2 and decreased activity of HA-degrading enzymes that overall cause massive expression of this incredibly high molecular-mass HA that ultimately confers protracted cancer protection [46]. Upon injury, moreover, HMW-HA is broken down into LMW-HA fragments that activate the innate immune response by engaging TLR-2 and by promoting Ag-specific T cell response in vivo [47]. In this context, HMW-HA may play a more homeostatic role. Although degradation products of HA have been found to initiate a proinflammatory response, their proposed binding to TLRs has long been controversial. There are reports showing direct binding of LMW-HA to TLR4 in murine dendritic-cells [48], supported by genetic evidence demonstrating the necessity of TLR4 for HA-evoked NF-κB response. A similar effect was observed by blocking TLR4 with a specific antibody in mouse chondrocytes exposed to LMW-HA fragments [49]. However, comparative analysis of other TLRs involved in the inflammatory response done by mutating the TLR2 receptor, failed to generate the same outcome [48]. Notably, others have shown lack of evidence for HA-mediated TLR4 activation, but shown described LMW-HA as a TLR2 agonist [47]. More recently, hyaluronan has been described as a regulator of TLR signaling by creating a barrier for receptor accessibility around the cells and not by directly binding to any TLRs [50]. Therefore, the debate is open and a clearer understanding of HA-evoked TLR signaling needs to further elucidated.

Several studies have shown that the CD44 expressed on the surface of malignant cells interacts with hyaluronan-rich microenvironment, thereby affecting cell-signaling pathways that induce migration, invasion of the basement membranes, and metastasis [51]. In cancer stem cells, HA/CD44 interactions evoke signals required for self-renewal and maintenance [52]. The survival and growth of cancer stem cells is partly explained by their resistance to oxidative stress conferred by CD44. Metabolic modulation by CD44 contributes to the antioxidant status of CSCs by maintaining low levels of ROS, therefore protecting the cells from ROS-induced cellular damage [52]. CD44 is also responsible for HMW-HA enhanchment of Ras signaling in ovarian tumor cells [53]. On the contrary, short hyaluronan oligomers can affect selective signaling pathways that boost immune response and change blood vessel growth counteracting tumor development [54]. Notably, both CD44 and RHAMM play pivotal roles during the metastatic process. Increased CD44 expression has been positively correlated to promoting breast cancer invasion and metastatic spreading to the liver [55] and, together with RHAMM, can induce endothelial cell proliferation and angiogenesis towards the metastatic lesions [33]. Thus, it is fully conceivable that targeting CD44/RHAMM could become an attractive new therapeutic approach against cancer.

Hyaluronan in cancer therapy

Potentially, all members of the HA signaling pathway represent promising novel anti-cancer therapy targets. The high clinical levels of their expression in a multitude of types of cancer make them ideal candidates. Treatments that interfere with the intracellular signaling responsible for tumor growth, invasion and angiogenesis such as the PI3-kinase/Akt, Erk, Ras, Src pathways, have been generated and include small-molecule inhibitors and antibodies. Anti-CD44 monoclonal antibodies have been developed to target CD44 for cancer therapy. In immunodeficient mice bearing MDA-MB-231 xenografts, treatment with anti-CD44 antibody resulted in activation and initiation of an immune response with consequent macrophage-mediated phagocytosis of the tumor cells [56]. In another study, RHAMM vaccines have been tested in clinical trials to combat specific forms of leukemia in patients with positive RHAMM expression on malignant cells. Vaccination with RHAMM-derived peptides in patients with malignant myeloid diseases caused a sustained immunological response with a significant increase of specific CD8+ T cells [57]. Finally, 4-methylumbelliferone (4-MU) is a well-established small molecule that targets HA synthesis and consequently inhibits cancer proliferation by lowering HA levels [58]. Orally dietary supplement of 4-MU showed onco-suppressive proprieties in breast cancer mediated by a decrease hyaluronan accumulation in the ECM, downregulation of HAS2 and concomitant upregulation of HYAL-1 and HYAL-2 [58]. Additional studies have shown that 4-MU inhibits HA synthesis not only by decreasing the levels of the enzymes involved with its synthesis but also by sequestering glucuronic acid, ultimately inhibiting proliferation, invasion, and migration in prostate, breast, ovarian and melanoma carcinomas [54]. Targeting the enzymes involved in HA synthesis (HASes) and the natural receptor is, therefore, promising in blocking cancer development and spreading and may be a potential strategy to fight cancer growth. However, more studies are needed to understand the molecular mechanisms by which different HA molecular weights act distinctly physiologically and in the biology of cancer progression and chemotherapy resistance.

Role of hyaluronan synthases in malignancy

As HA is a crucial factor able to favor all the hallmarks of cancers, understanding the mechanism of HA regulation in tumors is clinically relevant. There is mounting evidence supporting a causative link between upregulation of HASes and poor prognosis in cancer patients [51,59,60]. Mechanistically, many experiments on cancer-derived cell lines have highlighted the critical roles of HASes in supporting not only proliferation, survival, and motility but also chemo-resistance. The only known exception to this general evidence is cutaneous melanoma where poor prognosis was associated with decreased expression of HAS1 and HAS2 in the cancer cells vis-à-vis benign melanocytic lesions [61]. Contrary to other glycosaminoglycans that are synthesized in the Golgi, HA synthesis takes place at the plasma membrane, and it is catalyzed by three enzymes named HA synthase 1, 2, and 3 (HAS1/2/3) [20,22] that share about 60–70% identity at the amino acid level [62]. In vitro studies revealed that HAS2 and three possess higher enzymatic activity than HAS1, and HAS2 and HAS1 can synthesize an HA polymer of higher mass than HAS3 [63]. The precise role of each HAS isoforms in cancers or other physiological processes, is mostly unknown as there are no isoform-specific inhibitors of HAS activity (4-MU is the only known to inhibit HA synthesis) [64], and alternative HAS isoforms compensate for the loss/silencing of a specific HAS.

The majority of the published studies have focused on HAS2: the most important HAS isoform in development and the most abundant in adult tissues. Indeed, Has2^−/− mice are embryonic lethal [65,66]. Specifically, Has2^−/− embryos die between embryonic day 9.5 and 10.5 and exhibit severe cardiac and vascular abnormalities, in addition to yolk sac and somite deformities. Notably, some of the cardiac defects can be rescued in explants from Has2^−/− mice by exogenous HA [65,66]. Interestingly, knockdown of Has2 in zebrafish leads to abnormal blood vessels with static venous flow, suggesting that HAS2 might be involved in the pathogenesis of venous disesease as well [67].

During mouse development, HAS1 and HAS3 have restricted expression patterns [68], but their specific roles in adult tissues have not been adequately deciphered. Recently, HAS1 has been studied in detail [69], and it has been found associated with poor patient survival in ovarian cancer [70], colon cancer [71], Waldenström’s macroglobulinemia [72] and multiple myeloma [73]. HAS3 overexpression is linked to the formation of microvillus-like cell surface protrusions [74], which have been linked to increased release of hyaluronan-coated and plasma membrane-derived microvesicles [75]. HAS3 has also been described to play a role in pancreatic cancer [76] and to alter mitotic spindle and epithelial organization [77] promoting the malignant phenotype [78]. Interestingly, HAS3 seems to have a specific function in blood vessels in promoting atherosclerosis neointima formation [79]. Recently, the primary regulator of epithelial biology p63 isoform ΔNp63 was found to regulate HA metabolism by altering HAS3 and expression of hyaluronidases in the head and neck squamous cell carcinoma [80].

Several studies have demonstrated that HAS2 regulates tumor progression and cell aggressiveness [32,51,81,82]. For example, the triple-negative MDA-MB-231 and HS578T breast cancer cell line express higher levels of HAS2 mRNA and synthesize higher amounts of HA than breast cancer cell lines with a less aggressive phenotype, like MCF-7 [83]. Moreover, HAS2 is dramatically increased in bone metastases compared to the parental MDA-MB-231 cells, indicating that HAS2 expression has a pivotal role in cell motility and invasion. Similarly, mammary tumor biopsies in which HAS2 is overexpressed display enhanced angiogenesis and inflammatory cells recruitment [39]. In support of these findings, HAS2 suppression by RNA antisense or HA-synthesis inhibition by 4-MU reduces tumorigenesis and progression of breast cancer cells [84–86].

The tumor microenvironment is often enriched in growth factors, such as PDGF-BB and TGF-β, as well as tumor-promoting agents (phorbol 12-myristate 13-acetate) and glucocorticoids. In all cases, this enriched stroma can support HAS2 expression in tumor cells as well as stromal cells [87]. Interestingly, HAS2 is one of the primary enzymes regulated in cancer-associated fibroblasts [88] suggesting the possibility that tumor cells induce stromal cells to produce HA. In breast cancers, a high HA deposition in the stroma is related to lymph node positivity, poor differentiation, and poor patient survival [81]. HAS2 expression is unusually high in basal-like and triple negative tumors where HAS2 is a critical factor that induces epithelial-mesenchymal transition (EMT). HA and HAS2 have also been described to have crucial functions in determining cancer-initiating cells, or as cancer stem cells, that are believed to drive cancer growth and progression through aberrant self-renewal and generation of heterogeneous cancer cell lineages [21,89]. Collectively, we can propose a scenario where the presence of HA in the tumor microenvironment can affect not only cancer cell behavior but can also dramatically change other critical steps of tumorigenesis like immune cell infiltration and angiogenesis.

HAS2 regulation and cancer

The cellular machinery for HA synthesis uses the cytosolic precursors UDP-GlcUA and UDP-GlcNAc that have been recently described to be involved in modulating cancer HA synthesis as well as aggressiveness (Fig. 2) [90]. These two UDP-sugar nucleotides are synthesized by UDP-glucose dehydrogenase (UGDH) and by the hexosamine biosynthetic pathway (HBP), respectively. UGDH is a very interesting enzyme as it oxidizes UDP-glucose to form UDP-GlcUA with the production of two NADH molecules [59,91] and, therefore, altering the NAD: NADH ratio. Although NADH is critical for mitochondrial activity, altered NAD levels can dramatically affect cell metabolism as they can affect cyclic ADP-ribose, ADP-ribose, mono- or poly-ADP-ribosylation of proteins, and modulate sirtuins (NAD-dependent deacetylases) which have a central role in aging and are considered protective enzymes [92]. UDP-GlcUA is an essential metabolite as it is contained in almost all GAGs (except keratan sulfate) and it is the precursor for UDP-xylose, which is critical for the tetrasaccharide linkage region in proteoglycans synthesis [93]. Interestingly, UDP-GlcUA can also be used by glucuronyltransferase enzymes that belong to the class of detoxifying enzymes [94]. Therefore, an increase in UDP-GlcUA could drive increased HA synthesis as well as contribute to an enhanced chemoresistance, typical of aggressive cancers.

Fig. 2.

Schematic representation of the intracellular pathways that lead to the production of substrates of HAS enzymes and the role of HA in signaling. In the scheme are also indicated AMPK, O-GlcNAcylation and sirtuins that have a role in the regulation of metabolism and, therefore, can be altered by an active HA synthesis.

Tumors undergo a dramatic reprogramming of central metabolic pathways to obtain energy and build blocks to sustain cell growth and division [95]. UDP-GlcNAc is the final product of the hexosamine biosynthetic pathway, which is altered in several cancers [96]. Interestingly, UDP-GlcNAc is a critical factor for O-GlcNacylation of proteins, a post-translational modification that consists of the alteration of hydroxyl residues with GlcNac. This reversible modification of nuclear as well as cytoplasmic proteins is driven by O-GlcNAc transferases and removed by OGA [97]. Notably, O-GlcNAcylation has a dramatic impact on cancer cells as it contributes to several fundamental tumorigenic functions, including regulation of proliferative signaling, resistance to cell death, replicative immortality, and enhanced angiogenesis and invasiveness [96]. O-GlcNacylation can also regulate HA metabolism by increasing HAS2 stability at the plasma membrane, thus favoring HA deposition [91], as well as by controlling HAS3 localization and trafficking [98]. Interestingly, the intracellular trafficking of HASes in the secretory pathway can represent one of the major ways HA synthesis is regulated [99]. O-GlcNAcylation is not only a cytosolic process, but it also modifies nuclear proteins such as histones. Indeed, HAS2 promoter accessibility and transcriptional activity can be regulated by glucosamine precursors via O-GlcNacylation and by the natural antisense transcript HAS2-AS1, a member of the long non-coding RNA family [100].

Emerging studies from the human genome-sequencing project have shown that more than 80% of the human genome is actively transcribed into RNA, but just a little amount (3%) of the total RNA codes for translated proteins. Although non-coding RNA (ncRNAs) was initially considered a sort of “transcriptional noise” or a sequence artifact, recent studies have clearly shown that this portion of the transcriptome could play a key role in regulating gene expression [101]. Interestingly, in eukaryotes, the proportion of ncRNAs increases with the complexity of the organism, suggesting that these ncRNAs provide an extra layer of developmental complexity required for the evolution of eukaryotes [102]. In general, these ncRNAs include some of the classical housekeeping RNAs like ribosomal RNA (rRNA) and transfer RNA (tRNA). However, relying on transcript size, new classes of ncRNAs, with a regulatory function have been described: short ncRNAs characterized by <200 nt and long ncRNA which are >200 nt. Short ncRNA can be further classified into microRNAs (miRNAs), short interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA) and PIWI-interacting RNA (piRNA ) [103]. HAS2-AS1 is a tetra exonic natural antisense transcript synthesized by the opposite genomic DNA strand at the HAS2 locus, on human chromosome 8, which was first identified as lncRNA [85]. HAS2-AS1 overexpression can indeed suppress HAS2 expression and, thereby, HA production in human osteosarcoma cells [85]. Other studies demonstrated that HAS2-AS1 stabilized HAS2 mRNA in renal proximal tubular epithelial cells [104] and that HAS2-AS1 evokes chromatin remodeling around HAS2 promoter, ultimately favoring HAS2 transcription [100]. Recently, the coordinated expression of HAS2-AS1 and HAS2 was also described in oral squamous cell carcinoma, where HAS2-AS1-mediated hypoxia-induced cell invasiveness and EMT by stabilizing HAS2 [24]. Similarly, HAS2-AS1 is involved in TFG-β-induced EMT in breast cancer cells [105], and in papillary thyroid cancer recurrence [106]. Furthermore, HAS2-AS1 is present not only in the nucleus [100], but also in the cytosol [105]. Thus, the natural antisense transcripts can play other functions [107] by interacting with their own “sense” counterparts [101] or by competing with miRNA or can serve as sponge transcripts or ceRNAs (competing endogenous RNAs) [108] (Fig. 3).

Fig. 3.

Schematic representation of the genomic organization of human HAS2 locus on chromosome 8. Nuclear HAS2-AS1 can influence chromatin organization around HAS2 promoter regulatingHAS2 transcription, whereas cytosolic HAS2-AS1 can have different functions that can regulate HAS2 mRNA but also other processes interacting with other factors as miRNA.

The synthesis of HA requires energy, and it has been described that AMPK, the master cellular regulator of energy homeostasis, inhibits HAS2 activity through the phosphorylation of Thr110 [109]. The role of AMPK in the cell is to sense AMP increments, which are a clear indication of ATP depletion. Hence, AMPK activates catabolic processes aimed at restoring ATP levels and inhibiting ATP consuming pathways (i.e., anabolism). As several anabolic pathways are induced in malignancies, it is generally accepted that AMPK activation inhibits tumor proliferation. In support of this idea, there is clinical and epidemiologic evidence that patients treated with metformin (an antidiabetic drug that activates AMPK) show a reduced incidence of cancer [110]. As metformin reduces HAS2 activity by inducing HAS2 phosphorylation [109], the hypothetical anticancer effect of AMPK inducers could foresee not only the cellular blocking of anabolism but also influence the composition of the tumor microenvironment by reducing the critical pro-tumorigenic agent HA.

Concluding remarks

Cancer cells benefit from increased levels of the multifunctional hyaluronan that forms the structural basis of the pericellular and intercellular matrix. There is mounting evidence that during tissue remodeling evoked by cancer growth or inflammation, there is a new provisional matrix enriched in hyaluronan and its binding partners [111,112]. Hyaluronan is generated at the cell surface by a tri-member class of hyaluronan synthases capable of extruding this massive linear polysaccharide directly into the tumor microenvironment or to adjacent cell surface receptors, such as CD44 and RHAMM. This unique mode of synthesis and delivery makes hyaluronan an ideal factor in promoting angiogenesis and regulating the peri-neoplastic immune response. Obviously, minute alterations in either synthetic or degrading (hyaluronidase) enzymes could have profound effects on the milieu in which tumors need to grow and eventually metastasize. In addition, the numerous hyalectans, hyaluronan-binding proteoglycans, would add another layer of complexity given the fact that these proteoglycans are often differentially expressed in various malignancies and carry unique and variable sets of bound growth and pro-angiogenic factors. For example, the G3 domain of versican can promote tumor growth, and angiogenesis [113] and versican protects cells from oxidative stress-induced apoptosis [114]. Notably, overexpression of versican V3 promotes anti-inflammatory phenotypes [115] and concurrently stimulates mammary tumor growth and metastasis [116–118]. Thus, all of these factors should be considered when interpreting tumor stroma behavior and prognostic indicators from large patient populations. Hyaluronan, being at the crossroads of so many cellular pathways critical to cancer biology, needs to be investigated in depth. Hyaluronan presence, dimension, and strategic accumulation demands close scrutiny especially in understanding the molecular mechanisms by which different forms of hyaluronan affect cancer progression and chemoresistance.

Abbreviations

4-MU

4-methylumbelliferone

AMP

5’ adenosine monophosphate

AMPK

5’ adenosine monophosphate-activated protein kinase

ATP

adenosine triphosphate

BM

basal membrane

ECM

extracellular matrix

GAGs

glycosaminoglycans

GlcA

glucuronic acid

GlcNAc

N-acetylglucosamine

GLUT1

glucose transporter

HA

hyaluronic acid, hyaluronan

HAS1-3

hyaluronan synthases

HAS2-AS

hyaluronan synthases 2 antisense RNA1

HMW HA

high molecular weight hyaluronan

LMW HA

low molecular weight hyaluronan

NAD

nicotinamide adenine dinucleotide

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

PDGF-BB

Platelet-Derived Growth Factor-BB

PG

proteoglycans

PI3

phosphoinositide 3-kinase

RHAMM

hyaluronan-mediated motility receptor

RNA

ribonucleic acid

TGF-β

transforming growth factor β

TLR-2/4

toll-like receptor type 2/4

UDP-GlcNAc

uridine diphosphate N-acetylglucosamine

UDP-GlcUA

Uridine diphosphate glucuronate

UGDH UDP-

glucose 6-dehydrogenase