Hyaluronan: A modulator of the tumor microenvironment

Accepted Manuscript Title: Hyaluronan: a modulator of the tumor microenvironment Author: Theerawut Chanmee, Pawared Ontong, Naoki Itano PII: DOI: Reference:

S0304-3835(16)30099-4 http://dx.doi.org/doi: 10.1016/j.canlet.2016.02.031 CAN 12772

To appear in:

Cancer Letters

Received date: Revised date: Accepted date:

17-10-2015 16-2-2016 17-2-2016

Please cite this article as: Theerawut Chanmee, Pawared Ontong, Naoki Itano, Hyaluronan: a modulator of the tumor microenvironment, Cancer Letters (2016), http://dx.doi.org/doi: 10.1016/j.canlet.2016.02.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hyaluronan: a modulator of the tumor microenvironment Theerawut Chanmeea, Pawared Ontongb, Naoki Itanoa,b,c,* a

Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University,

Kita-ku, Kyoto, Japan b

Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo

University, Kita-ku, Kyoto, Japan c

Institute of Advanced Technology, Kyoto Sangyo University, Kita-ku, Kyoto, Japan

*Corresponding author. Tel.: +81-75-705-3064; Fax: +81-75-705-3064. E-mail address: [email protected] (N. Itano)

Highlights 1. The HA-rich tumor microenvironment recruits and activates stromal cells to enhance tumorigenicity. 2. Tumor-derived and stromal-derived HA molecules remodel tumor microenvironment to promote tumor progression. 3. HA regulates cancer stemness through the induction of EMT.

ABSTRACT Tumors are cellular masses formed through dynamic interactions between tumor cells and a mixed population of stromal cells. Crosstalk between oncogenic and adjacent stromal cells contributes to the formation of a “tumor microenvironment” influencing the tumor cell behaviors of proliferation, invasion, and metastatic spread throughout cancer progression. The composition and structure of the tumor microenvironment vary among different types of tumors and are extensively

remodeled

in

close

association

with

tumor

advancement.

The

tumor

microenvironment is composed of not only cellular compartments, such as endothelial cells, fibroblasts, inflammatory cells, and immune cells, but also of bioactive substances, including growth factors and the extracellular matrix. Hyaluronan (HA) is a major component of the 1

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extracellular matrix, and the degree of HA accumulation is strongly correlated with a poor prognosis in advanced cancer patients. Emerging evidence has suggested that HA creates a specific microenvironment that is favorable for tumor angiogenesis, invasion, and metastasis. This review highlights the prominent roles of HA as a modulator of the tumor microenvironment and addresses the recent advances regarding HA function in cancer stem cell niches.

Keywords: Hyaluronan; Tumor microenvironment; Epithelial-mesenchymal transition; Cancer stem cell; Stromal cells

Introduction The tumor microenvironment is widely recognized as a key player in the promotion of tumor growth and malignant progression. It is composed of heterogeneous cellular and non-cellular compartments and is extensively remodeled during tumor advancement [1,2]. The tumor microenvironment contains multiple types of stromal cells, including tumor-associated macrophages (TAMs), tumor-associated fibroblasts (TAFs), mesenchymal stem cells (MSCs), and endothelial cells. Interactions between tumor and stromal cells play a critical role in tumor initiation and progression. Over the past decade, considerable efforts have been made in exploring how these stromal cells facilitate tumor initiation and progression. In concert with malignant cells, stromal cells play prominent roles in the formation and modulation of the tumor microenvironment by producing a wide variety of soluble signaling molecules [3,4]. Moreover, the significance of extracellular matrix (ECM) components in tumor development has also been highlighted by pathological and experimental studies [5-7]. Aberrant ECM turnover and organization are often detected in tumor and appear to be involved in many biological aspects supporting tumor progression. There is growing evidence that certain ECM components have pro-angiogenic, pro-inflammatory, and anti-apoptotic effects. Among such molecules, tenascin-C [8], biglycan [9], and versican [10] have all been demonstrated to stimulate innate immune responses and thereby establish a pro-inflammatory microenvironment for tumor growth. Increased ECM rigidity from the accumulation of collagen 2

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crosslinking also promotes integrin clustering and enhances the cellular signals that regulate tumor cell survival, proliferation, and invasion [7]. Moreover, ECM degradation gives rise to the generation of bioactive fragments which act directly as inflammatory stimuli. For instance, proteolytic cleavage of type IV collagen generated a cryptic epitope required for angiogenesis and tumor growth [11]. As an integral component of the ECM, hyaluronan (HA) influences the fundamental aspects of cellular biology through both direct and indirect actions. HA mediates many cellular events during embryonic morphogenesis, cellular regeneration, and wound healing, while abnormalities in HA have been implicated in many diseases, such as inflammatory disorders and cancer [12]. In the tumor microenvironment, HA provides a three-dimensional scaffold for cells by the assembly of pericellular ECM and regulates the behaviors of stromal and tumor cells, in some cases through interactions with HA receptors. A wide variety of HA-binding molecules contributes to the assembly of pericellular ECM and specifically regulates a diverse array of HA functions. Although HA is a polysaccharide of a relatively simple composition, it exhibits multiple properties dependent largely on its molecular size and tissue concentration, both of which are firmly regulated by the concerted activities of biosynthetic and degradation processes [13]. In this review, we offer important insights into the roles of HA amid the crosstalk between malignant and tumor-associated stromal cells and describe how HA modulates the tumor microenvironment to promote tumor development and progression.

Abnormal HA synthesis and degradation in tumors

HA is a large polysaccharide composed of repeating N-acetylglucosamine and glucuronic acid disaccharide units (Fig. 1A). Under normal physiological conditions, HA can be composed of up to 25,000 disaccharide units, but these chains become smaller and more dispersed in a pathological status [13]. It is now accepted that the biophysical functions of HA vary depending on its molecular size. For instance, low molecular weight HA (LMW-HA) of a defined size (generally <200 kDa) is able to induce inflammation and angiogenesis [14], while high molecular weight HA (HMW-HA) is anti-angiogenic [15] as well as immunosuppressive [16]. 3

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The molecular size of HA is firmly regulated by the concerted activities of biosynthetic and degradation processes. In mammalian cells, each of three HA synthases (HAS1-3) possessing different enzymatic properties synthesize HA by repeated addition of each monosaccharide component to the nascent HA chain and extrude it through the plasma membrane to the cell surface or into the ECM [17] (Fig. 1B). HA catabolism is controlled by a family of endo-N-acetylhexosaminidases called hyaluronidases. Six different hyaluronidase (HYAL1, 2, 3, 4, HYALP1, and PH-20) genes have been identified in humans [18] whose expression is either tissue-specific or responsive to microenvironmental stimuli. HYAL1 and HYAL2 are the major hyaluronidases expressed in most tissues and hydrolyze the β (1-4) glycosidic linkage between N-acetylglucosamine and glucuronic acid to produce HA fragments of varying sizes. Mechanistically, HMW-HA is tethered to the cell surface by CD44 in caveolin-rich lipid rafts and cleaved by glycosylphosphatidylinositol (GPI)-anchored HYAL2 [19]. HYAL2 generates intermediate sized HA fragments that are eventually delivered to endo-lysosome compartments, wherein HYAL1 and lysosomal β-exoglycosidase enzymes further degrade the fragments into oligosaccharides [20] (Fig. 1C). Recently, KIAA1199 has been shown to play a central role in HA degradation in a manner independent of CD44 and HYAL [21]. This molecule was originally described as an inner ear-specific protein involved in non-syndromic hearing loss [22]. Cell surface KIAA1199 can bind to HA and internalize the sugar chain via clathrin-coated pits. Blockage of either the internalization or knockdown of KIAA1199 suppressed the fragmentation of internalized HA [21], suggesting that the HA catabolism mediated by this molecule occurred through vesicle endocytosis (Fig. 1C). Accumulation of HA and its function in tumor are rather complex. Within tumor, HA is abundantly synthesized by both cancer and stromal cells and the high level of HA has been reported to be closely associated with tumor aggressiveness. Clinicopathological analyses have demonstrated that accumulation of HA in malignant breast [23], prostate [24,25], ovarian [26], and lung [27] cancers are strongly associated with tumor aggressiveness and a poor disease outcome. Although widespread evidence supports the central role of HA in many aspects of tumor progression, contradictory observations have also been made in both clinical and experimental studies. Differing HA levels were found in oral squamous cell carcinoma patients, 4

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in whom reduced HA was associated with poor survival [28]. Likewise, decreased levels of HA and CD44 were linked to an unfavorable prognosis in clinical stage I cutaneous melanoma [29], and an extremely high level of HA inhibited tumor growth [30]. These observations therefore suggest that HA may regulate tumor aggressiveness positively as well as negatively in a contextdependent manner. The dynamic balance between concurrent HA synthesis and catabolism may be a prominent factor in tumor advancement.

HAS expression in both stromal and malignant cells has been shown to relate to tumor aggressiveness and low cancer patient survival rates [31]. Dysregulation of HAS1 alternative splicing that generated truncated variants, such as HAS1V, was correlated with significantly reduced survival in multiple myeloma patients [32]. The prominent role of HA in tumor progression has been supported by experimental studies, wherein increased HA production by forced expression of HAS1, HAS2, or HAS3 increased tumor growth and metastasis in xenograft models of breast cancer [33], prostate cancer [34], and fibrosarcoma [35]. Has2 overexpression experiments in a mouse model of breast cancer have provided further evidence on the importance of HA in tumor progression. HA overproduction induced marked stromal induction and accumulation of HA within tumor stroma. Concurrent with these events, blood and lymphatic vessels were formed in nearby stroma surrounding tumor cell islets [6,36], and the mechanism of HA-mediated stromal cell recruitment was found to rely on the presence of versican [6]. Correspondingly, a reduction in HAS2 inhibited the tumorigenesis and progression of breast cancer [37], and impaired tumor growth was observed when interactions between HA and tumor cells were disrupted [38]. The accumulation of LMW-HA has also been associated with tumor aggressiveness [39-41] in that HA oligosaccharides possess the ability to trigger the expression of specific cytokines and proteases necessary for ECM remodeling. For instance, LMW-HA of 4-6 oligomers in size enhanced MMP2 and IL-8 expression in human melanoma cells via toll-like receptor (TLR) 4 and subsequent activation of NF-κB [42]. Interactions of LMW-HA with CD44 and TLR signaling through MyD88/NF-κB led to the production of IL-1β/IL-8 and increased invasiveness in breast cancer cells [43]. Furthermore, LMW-HA-induced innate immune responses may enhance tumor growth and metastasis [44]. Consistent with the accumulation of LMW-HA, 5

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hyaluronidase levels are often increased in several types of cancers, such as those of colon [45], bladder [46], prostate [47], brain [48], and breast [49], and its induction has been linked to tumor grade and metastatic spread. KIAA1199 has also been highlighted in tumor progression and invasiveness due to its high expression in several aggressive cancers, such as those of the breast [50], prostate [51], and colon [52]. Conversely, decreased expression of hyaluronidases has also been reported in certain tumors such as head and neck squamous cell carcinoma [53], endometrial [54], and pancreatic [55] cancer. For instance, decreased expression of HYAL1 was correlated with poor survival in pancreatic cancer patients [55], while hyaluronidase treatment reduced the growth of tumor xenograft [56]. In light of this, abnormal synthesis and degradation of HA in advanced tumors may facilitate tumor cell aggressiveness by both accumulation of HA and generation of HA oligosaccharides.

HA-dependent changes in stromal cell behavior and modulation of the tumor microenvironment Tumor-associated macrophages TAMs are important stromal cells in tumors that play crucial roles in driving growth and progression. The well documented pro-tumor activities of TAMs include stimulation of tumor cell proliferation and migration, promotion of angiogenesis, immunosuppression, and microenvironment remodeling [57,58]. Clinicopathological studies have also highlighted a link between an abundance of TAMs and tumor aggressiveness [59,60]. In contrast, several studies have implicated that the presence of macrophages in tumors improved patient prognosis. Prominent macrophage infiltration tended to positively influence prognosis in colon cancer patients [61]. Moreover, the accumulation of macrophages in prostate [62] and colorectal cancers [63] was associated with higher survival. Pathological evidence has demonstrated that these macrophages, each with a distinct phenotype, exhibit significant heterogeneity in their localization pattern, implying that the differentiation of infiltrating monocytes is influenced by the local microenvironments within tumor masses. For example, macrophages in the peritumoral stroma markedly expressed major histocompatibility complex (MHC) class II, which suggested that they were newly infiltrating anti-tumor macrophages. In contrast, macrophages in cancer 6

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nests were negative for MHC class II and expressed anti-inflammatory cytokine IL-10, indicating that they represented pro-tumor phenotype [64]. The mechanisms underlying monocyte recruitment and differentiation in tumor masses have been investigated. Once circulating monocytes guided by tumor-derived chemoattractants lodge within tumors, they begin differentiation in response to local microenvironmental stimuli. Several cytokines, chemokines, and growth factors, including granulocyte-macrophage colonystimulating factor, macrophage colony-stimulating factor, chemokines CCL2, CXCL10, and CXCL12, and transforming growth factor-β (TGF-β), collectively orchestrate the multiple phases of monocyte recruitment, monocyte-to-macrophage differentiation, and macrophage activation [65]. There is also growing evidence that ECM molecules can activate macrophages as well (Fig. 2A). Among such molecules, HA and its binding proteoglycans have been demonstrated to initiate TLR-mediated innate immune responses. Versican, a large chondroitin sulfate proteoglycan, is involved in ECM assembly via associations with HA and various other ECM constituents. Kim et al. recently uncovered a new inflammatory pathway in which versican activated macrophages by ligation of TLR2 and elicited the production of pro-inflammatory cytokines [66]. As a major ECM component, HA has frequently been implicated in monocyte/macrophage trafficking and activation. Pathological studies of human breast cancer specimens suggested that increased numbers of macrophages were correlated with HA accumulation in tumors [67]. We have also demonstrated that TAMs preferentially infiltrate into mammary tumors in a manner dependent

on

an

HA-rich

tumor

microenvironment

and

concomitantly

enhance

neovascularization and tumor growth [36]. Mechanistically, these phenomena may be due to the concerted action of HA and its binding molecules. Pericellular HA cable-like structures, composed of inter-α-inhibitor heavy chains, TNF-stimulated gene-6 (TSG-6), and versican [68], provide a suitable scaffold for the mobilization of mononuclear leukocytes [69] (Fig. 2A). Upon binding to the cable-like framework via cell surface CD44, peripheral blood monocytes are activated to produce growth factors and ECM components. Meanwhile, HA degradation products also activate monocytes/macrophages to induce the specific gene expression programs required for inflammation and ECM remodeling. For instance, HA oligosaccharides utilized both TLR2 7

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and TLR4 as receptors to stimulate inflammatory gene expression in macrophages and act as an endogenous danger signal [70,71]. Macrophages polarize into diverse subsets, schematically identified as M1 (i.e., classically activated) and M2 (i.e., alternatively activated). M1 macrophages function in host defense mechanisms against bacteria and viruses and anti-tumor immunity, while M2 macrophages are widely termed as anti-inflammatory, immunoregulatory, and pro-tumor cells [72]. The polarization among distinct macrophage states is regulated in response to a wide variety of signals in the local microenvironment. In this regard, HA oligosaccharides have been shown to induce early activation of monocytes and subsequent development of immunosuppressive macrophages [64]. This view is consistent with the finding that, through CD44 and TLR-4, HA oligosaccharides downregulated the expression of inflammatory cytokines TNF-α and IL-12 and upregulated the expression of Th2 cytokines IL-10 [73] (Fig. 2A). Earlier studies have reported that HA oligosaccharides act on monocytes and macrophages to drive them towards an M2 subtype [64]. The differentiated M2 macrophages then start to produce Th2 cytokines and several angiogenic factors, leading to the enhancement of tumorigenicity through their immunosuppressive and pro-angiogenic actions.

Tumor-associated fibroblasts

HA facilitates fibroblast recruitment by modulating the tumor microenvironment Fibroblasts residing in the tumor margin or infiltrating into the tumor mass, termed TAFs, are the major cellular stromal components in many solid tumors [74-76]. TAFs are phenotypically distinct from normal fibroblasts and display the myofibroblast-like characteristics of rapid proliferation rate and expression of mesenchyme-specific proteins, such as α-smooth muscle actin (α-SMA), fibroblast-specific protein-1 (S100A4), fibroblast-activating protein, and vimentin [74,77]. There is conflicting evidence on the role of TAFs in tumor formation and growth: TAFs exhibit pro-tumor activity in certain types of cancers while act as tumor suppressors in others. TAFs contribute to tumor promotion by actively secreting a wide spectrum of growth factors, chemokines, and cytokines [78]. TAF-secreted hepatocyte growth factor (HGF) directly enhanced the proliferation and survival of cancer cells [79]. Paracrine signaling 8

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of TAF-derived factors, such as CCL2 and vascular endothelial growth factor (VEGF), has also been proposed to contribute to tumor formation and progression through the recruitment of macrophages and endothelial cell progenitors [80-82]. Furthermore, TAFs produce both ECM components and its degrading enzymes, thereby involving themselves in the remodeling of the ECM and generation of a tumor microenvironment favorable for tumor cell proliferation and invasion. On the other hand, Chang et al. demonstrated that TAFs expressing Slit2 could either suppress or promote the tumorigenicity of breast cancer cells [83]. These observations suggest that TAFs may regulate tumor growth positively as well as negatively in a context-dependent manner. Tumors can recruit fibroblasts and reprogram them into activated TAFs. Several lines of evidence have shown that tumor cells frequently drive fibroblast recruitment by tumor-derived growth factors, such as TGF-β and platelet-derived growth factor [84,85]. Dynamic changes in the ECM have been reported to promote fibroblast recruitment as well. Our earlier study using a transgenic mouse model demonstrated that HA overproduction in spontaneous mammary tumors led to an accumulation of TAFs in tumor stroma [6]. HA evokes fibroblast motility by acting on intracellular signaling pathways through interactions with its cell surface receptors. Specifically, the molecule may promote an invasive phenotype of myofibroblast via binding with CD44 since a CD44 deficiency attenuated the HA-dependent invasiveness of myofibroblasts [86]. This observation was consistent with the fact that 6-mer HA oligosaccharides were essential for ligand recognition by CD44 to stimulate fibroblast migration [87]. Such an effect was abrogated in mice deficient for receptor for HA-mediated motility (RHAMM) and CD44, suggesting that these receptors were required for a fibroblast response to HA oligosaccharides. It is also noteworthy that HA oligosaccharide-stimulated fibroblast migration and invasion required RHAMM to sustain CD44-mediated ERK1/2 activity [88]. Alternatively, HA accumulation in tumors may provide a microenvironment amenable to easy fibroblast penetration by increasing the turgidity and hydration of the ECM. The construction of an HA-rich ECM followed by the recruitment of fibroblasts might simultaneously drive tumor microenvironment remodeling through the deposition of other ECM components. For instance, TAF-derived collagen may generate a crosslinked network that contributes directly to ECM stiffness, thereby increasing cell adhesion and invasion [7]. 9

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Involvement of HA in TAF precursors Regarding the origin of TAFs, several possible sources have been proposed: (1) local fibroblasts or tissue-resident fibroblast precursors infiltrating into the growing tumor, (2) MSCs recruited from the circulation, (3) tumor cells undergoing epithelial-mesenchymal transition (EMT) in the tumor parenchyma, and (4) endothelial cells that undergo endothelial to mesenchymal transition (EndMT) (Fig. 2B). The most direct route is tissue-resident fibroblasts that transdifferentiate toward myofibroblasts by tumor-derived factors during their recruitment. Among the factors, TGF-β induces fibroblast differentiation through two cooperative signaling cascades: HA/CD44 and the epidermal growth factor receptor (EGFR)/ERK1/2 signaling. Midgley et al. illustrated how HA promoted TGF-β-driven fibroblast differentiation, describing that the assembly of HA pericellular coats promoted co-localization of CD44 and EGFR within lipid rafts and subsequent activation of the MAPK/ERK signaling pathway. This pathway is synergistic with that of TGF-β/Smad2, which is necessary for fibroblast differentiation [89]. Studies employing mouse models of inflammation-induced tumors have supported the premise that MSCs are a source of TAFs. Quante et al. reported that 20% of TAFs were derived from MSCs in a murine model of inflammation-induced gastric cancer [90]. The processes controlling MSC mobilization have been linked to HA-CD44 interactions, whereby HA cablelike structures might provide a scaffold for CD44-positive MSCs and facilitate their invasion into tumors. Indeed, CD44-deficient MSCs exhibited reduced infiltration into tumor stroma and a hampered ability to differentiate into myofibroblasts in a transgenic murine cancer model [91]. Lastly, myofibroblasts may be derived from cancer cells that undergo EMT. As HA overproduction induces EMT in normal epithelial cells [92,93], HA-dependent EMT might similarly convert tumor cells to TAFs and thereby function as a mechanism for TAF supply.

Endothelial cells and angiogenesis

Angiogenesis is an essential process in tumor growth and metastasis that requires specific sequential cellular events, such as endothelial cell activation and degradation of the basement membrane followed by endothelial cell migration and invasion into the stroma [94]. In addition 10

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to malignant cells, inflammatory cells, stromal fibroblasts, perivascular cells, and other tumorassociated stromal cells contribute significantly to the production of angiogenic factors. These factors trigger an angiogenic cascade accompanied by the dissociation of pericytes from blood vessels, degradation of the basement membrane, and proliferation and migration of endothelial cells [95]. Among such angiogenic factors, VEGF and basic fibroblast growth factor (bFGF) have emerged as central regulators [94]. The ECM surrounding the vasculature can also affect angiogenesis, either positively or negatively [77]. The ECM modulates tubular network formation and thereby influences angiogenesis through multiple mechanisms involved in regulating endothelial cell behaviors. The ECM surrounding normal vasculature is mainly composed of fibronectin, laminin, collagen, and proteoglycans [96]. On the other hand, in vivo studies have shown that ECM-derived HA oligosaccharides of 16 disaccharide units have the potential to induce pathological angiogenesis [6]. LMW-HA has long been recognized as a pro-angiogenic factor, and HA oligosaccharides along with VEGF synergistically stimulated endothelial cell proliferation, migration, and capillary formation [97]. Mechanistically, HA fragments can trigger the MAPK cascade via interactions with CD44 [98]. Furthermore, Lennon et al. found that LMW-HA induced the formation of CD44v10/EphA2 complexes and Src-mediated EphA2 phosphorylation, both of which are required for angiogenesis [99]. Microvascular endothelial cells express high levels of CD44 and HYAL2. Knockdown of either abrogated the formation of tubular networks, suggesting that angiogenic modulation was mediated by the binding of HA degradation products to CD44 [100]. RHAMM is also a receptor for HA that is expressed both on the cell surface and within intracellular compartments. Several studies have demonstrated RHAMM expression in endothelial cells and its role in angiogenesis modulation [101,102]. Since knockdown of either receptor was sufficient to inhibit LMW-HA-induced vessel formation [102], the HA receptors CD44 and RHAMM appear to be critical for new blood vessel formation; CD44 is the major receptor for endothelial cell adhesion and proliferation, while RHAMM is essential for endothelial cell invasion [101]. It is believed that CD44 and RHAMM activate distinct cascades in response to LMW-HA since CD44 mediated signaling through PKC-α and γ-adducin whereas RHAMM did not [102]. Meanwhile, HMW native HA has been considered to be anti-angiogenic because it inhibited endothelial cell proliferation and migration as well as capillary formation in a three-dimensional 11

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matrix in vitro [15], although recent in vivo studies suggested that HMW-HA might be angiogenic. Koyama et al. analyzed the molecular basis of HMW-HA-induced angiogenic promotion in vivo and witnessed a cooperative action between HA and versican in the mobilization of stromal cells and subsequent enhancement of bFGF-induced angiogenesis [6]. Another report described how HMW-HA augmented CXCL12/CXCR4 signaling associated with CD44 to facilitate vessel formation and angiogenesis [103]. Thus, the pro-angiogenic function of HMW-HA is likely due to the enhancement of signals induced by chemokines and growth factors. Overall, the balance of regulatory HMW-HA and effector HA oligosaccharides may figure prominently in promoting angiogenic responses (Fig. 2C).

Interstitial fluid pressure and vasculature

The interstitial fluid is a medium for the transport of nutrients and waste products between cells and the vascular system, and transcapillary flow into interstitial spaces is controlled by hydrostatic and colloid osmotic interstitial fluid pressure (IFP). IFP is very low in normal tissue, but can become markedly elevated in solid tumors [104]. Altered IFP has been disclosed in several types of tumors, such as breast [105], melanoma [106], colon [105], head and neck [107], and pancreatic ductal adenocarcinoma (PDA) [108]. A number of studies have suggested that high IFP can limit the efficacy of cancer treatment. The chemotherapeutic drugs and monoclonal antibodies used in cancer treatment are normally transported to interstitial spaces via pressure gradients. Increased IFP hampers the transport of these macromolecules, thus rendering therapy less effective. For instance, IFP often ranges from 75 mmHg to 130 mmHg in PDA, in stark contrast to 8 mmHg in the normal pancreas and -2.0 mmHg in muscle [109]. Along with its unique biophysical properties of strong water retention, HA can form a hydrogel-like ECM that exerts strong turgor force, and is thus likely involved in the mechanism for increased tumor IFP. In fact, HA level was correlated with IFP elevation in several reports, and systemic administration of HA degradation enzymes periodically lowered IFP and raised tumor cell susceptibility to cytotoxic chemotherapeutic agents [109-111]. Therefore, targeted disruption of HA polymers may alter a hydrated ECM and ameliorate the transport of chemotherapeutic agents.

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Roles of HA in EMT EMT is a cellular program that allows a polarized epithelial cell to acquire mesenchymal features. This process has been accepted as an essential developmental phenomenon that controls many embryonic events, such as gastrulation and neural crest formation [112]. Cells undergoing EMT are characterized by profound morphological changes from a cobblestone-like epithelial form to a spindle shape. During the EMT process, epithelial cells lose their cell-cell adhesion properties and apical-basal polarity and acquire a fibroblastic motile phenotype (Fig. 3A). Hallmarks of EMT are the downregulation of E-cadherin and upregulation of vimentin, both of which are tightly controlled by multiple signaling pathways. TGF-β is one of the most prominent EMT inductors, promoting the conversion of epithelial to mesenchymal features by transcriptional and post-transcriptional regulation of a distinct set of transcription factors [113]. A variety of such factors, including the zinc finger Snail homologues (Snail1 and Snail2/Slug) and several basic helix-loop-helix factors (Twist, ZEB-1, and ZEB2), have been demonstrated to promote EMT through the coordinated modulation of EMT-related genes [114]. An increasing number of studies are implicating EMT in tumor progression and metastasis. During tumor advancement, EMT produces many morphological and molecular alterations in tumor cells that are similar to those in the developmental EMT program. Tumor cells undergoing EMT have increased invasiveness via an enhancement in cell motility, reductions in cell-cell adhesion and cell polarity, and matrix remodeling [115]. Multiple extracellular stimuli, including HGF, EGF, platelet-derived growth factor (PDGF), Wnt, Notch, and TGF-β combine to orchestrate the EMT-related process by integrated networks of signal transduction pathways and transcription factors [114]. HA has also been associated with the acceleration of EMT. Elevated HA production in MCF-10A human mammary epithelial cells induced mesenchymal characteristics, including upregulation of vimentin, dispersion of cytokeratin, and loss of cell adhesion at intercellular boundaries [92]. Increased levels of HA further promoted anchorage-independent growth and invasiveness by stimulating the PI3K/Akt signaling pathway [92] and inducing MMP-2 and MMP-9 expression, respectively [116] (Fig. 3B). Perturbation of HA-receptor interactions by treatment with HA oligosaccharides reversed the invasiveness induced by HGF in MCF-10A 13

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cells, suggesting the involvement of HA receptors in EMT [92]. Indeed, CD44 interacts with ERM proteins (ezrin/radixin/moesin) in response to HA, which directly mediate cytoskeletal remodeling and promote migration and invasion [117,118] (Fig. 3B). Furthermore, HA-CD44 communication evoked nuclear translocation of the CD44 intracellular domain followed by expression of the EMT-related Twist transcription factor in human breast cancer [119]. Porsch et al. demonstrated that HAS2 knockdown inhibited both HA production and TGF-β-induced EMT and completely abolished TGF-β-accelerated cell migration in NMuMG mammary epithelial cells. However, inhibition of HA-CD44 binding by neutralizing antibodies did not abrogate TGF-β-induced EMT, suggesting a CD44-independent action of HA [120]. Our recent study with a murine mammary tumor model also showed in vivo that forced Has2 expression and resultant HA overproduction in tumor cells caused the loss of E-cadherin at cell-cell junctions and increased nuclear translocation of β-catenin [6]. Several studies have suggested that βcatenin contributes to the EMT program by inducing the expression of Slug [121] and Twist [122]. Although the significance of HA-CD44 interactions in EMT is controversial, these reports highlight the importance of HA in the dynamic regulation of the EMT program. Along with EMT, its reverse process, mesenchymal-epithelial transition (MET) may also play a critical role in a series of metastatic steps. Epithelial-mesenchymal plasticity, or the interplay between EMT and MET, is tightly regulated by environmental conditions and signals. HA has been implicated in the metastatic spread of various cancer types. Experimentally, we demonstrated that mutant mammary carcinoma cells lacking the ability to synthesize HA displayed a significant reduction in experimental lung metastasis, while restoration of HA synthesis by HAS1 transfection recovered the metastatic properties in accordance with the formation of HA pericellular coats [33]. An inhibitor of HA synthesis, 4-methylumbelliferone (4MU), inhibited liver metastasis of B16F-10 melanoma cells [123] and lung metastasis of osteosarcoma cells [124]. CD44 is frequently overexpressed in metastatic cancer cells and has been linked to the metastatic spread of numerous cancer types [125-127]. CD44 induction in the weakly metastatic MCF7 breast cancer cell line enhanced metastasis to the liver when injected into immunodeficient mice [128]. Meanwhile, absence of the CD44 gene prevented lung metastasis of osteosarcoma in mice with a tm1 mutation in the p53 gene [129]. Although it

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remains to be confirmed, the switch between EMT and MET may be instrumental in the HA/CD44-dependent acceleration of metastatic spread.

HA and cancer stem cell niches Stem cells reside in a special microenvironment called a “stem cell niche” that provides the major cues for promoting survival and maintenance. Stem cell behavior is influenced by direct interactions not only with neighboring stromal cells, but also with niche components, such as secreted factors, ECM molecules, hypoxia, and other environmental signals. A recent study has indicated that HA is a niche element required for bone marrow hematopoiesis. Human bone marrow mesenchymal stem cells typically synthesize a large amount of HA to retain their pericellular matrix and maintain stemness. HA depletion reduced the ability of the microenvironment to support these hematopoietic stem cells [130]. Similarly, inhibition of HA synthesis by 4-MU treatment eliminated hematopoiesis in long-term bone marrow cultures [131]. Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells that drive tumor initiation and progression [132,133]. Comparably to a normal stem cell niche, a CSC niche regulates the maintenance and expansion of CSCs. Recent investigations have uncovered certain extra- and intracellular signals that can revert cancer progenitors to selfrenewing and multipotent CSC states [134-137]. Thus, remodeling of the CSC niche is now thought to be a crucial step in tumorigenesis and tumor progression. An HA-rich ECM may provide a microenvironment favorable for the self-renewal and maintenance of CSCs. Okuda et al. reported that HA produced by metastatic breast cancers promoted interactions between CSCs and TAMs, which then secreted PDGF to activate fibroblasts and osteoblasts and support CSC self-renewal [138]. In human head and neck squamous cell cancer, binding of HA and CD44 variant form 3 propagated CSC stemness by inducing complex formation of the stem cellspecific transcription factors Oct4, Sox2, and Nanog [139]. Furthermore, HA/CD44-mediated Nanog activation promoted the expression of the stem cell regulators Rex1 and Sox2 [140] (Fig. 3C). CD44 knockdown could attenuate the expression of Oct4, Nanog, Sox2, and other stem cell markers [141]. Since stem cell-specific transcription factors are necessary for the development 15

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and maintenance of CSCs, these lines of evidence emphasize that both HA and CD44 are indispensable factors in CSC self-renewal. EMT is believed to mediate the reversion of cancer cells to CSCs. In support of this, induction of EMT in breast cancer cells by ectopic expression of Snail and Twist or prolonged TGF-β treatment stimulated the acquisition of stem cell properties [142]. Forced expression of Slug, a member of the Snail family, in collaboration with Sox9 efficiently induced the entrance of breast cancer cells into a CSC state [143]. Evidence supporting a key role of HA in EMT induction has also implicated an association of HA with the acquisition of CSC signatures. In our recent study, HA-overproducing mammary tumor cells acquired stemness via the upregulation of TGF-β and induction of Snail and Twist, while a loss of EMT by inhibition of TGF-β-Snail signaling or Twist knockdown markedly reduced CSC subpopulations [93]. CSCs are inherently resistant to cytotoxic chemo- and radiotherapy, thereby abrogating complete therapeutic elimination of tumor masses. The acquisition of multidrug resistance (MDR) in CSCs is mediated by the induction of survival/anti-apoptotic signals and increased expression of multidrug transporters and MDR genes. HA-CD44 binding elicited MDR by promoting expression of MDR1 and multidrug resistance protein 2 (Fig. 3C). These interactions also promoted Nanog phosphorylation and its nuclear translocation, which eventually resulted in the upregulation of MDR1 [144]. CSCs effectively gain a survival/growth advantage under oxidative stress conditions by adapting their metabolism and maintaining cellular redox homeostasis. Metabolic modulation by CD44 has been demonstrated to contribute to antioxidant status in tumor cells; CD44 caused metabolic reprograming from mitochondrial respiration to glycolysis in p53-deficient or hypoxic cancer cells by interacting with pyruvate kinase M2 [145]. CD44 variant form (CD44v8-10) also modulated defense against reactive oxygen species by altering cellular glutathione synthesis [146]. HMW-HA has been shown to decrease oxidative DNA damage in human corneal epithelial cells [147], and HA exerted protective effects against mitochondrial DNA damage, mitochondrial dysfunction, and mitochondria-driven apoptosis under conditions of oxidative injury [148].

Conclusions and future prospects 16

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This review emphasizes prominent roles of HA as a potent modulator of the tumor microenvironment. HA creates specific circumstances that are favorable for interactions between tumor cells and stromal cells to elicit a wide range of extracellular stimuli for tumor growth, angiogenesis, invasion, and metastasis. Recent evidence further suggests that HA serves as a niche to regulate CSC maintenance, propagation, and chemotherapeutic resistance. Thus, HA appears to have a significant impact on many aspects of tumor initiation and malignancy. A more complete understanding of the mechanisms underlying HA-driven modulation of the tumor microenvironment will facilitate the development of future anticancer treatments. Additionally, targeting of HA in the CSC niche in combination with conventional chemotherapy may provide a promising therapeutic approach for achieving successful cancer eradication.

Acknowledgments This work was funded by grants from JSPS KAKENHI Grant Number 26430125 (to N.I.); Kyoto Sangyo University Research Grant Number C1301 (to N.I.) and Tokyo Biochemical Research Foundation postdoctoral fellowships for Asian researchers in Japan (to T.C.).

Conflict of interest The authors have no conflicts of interest to disclose.

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Figure Legends Fig. 1. HA biosynthesis and degradation. (A) Structure of HA. HA is a negatively charged polysaccharide composed of repeating disaccharide units of glucuronic acid and Nacetylglucosamine. (B) HA is synthesized at the plasma membrane by HAS1-3. HAS enzymes catalyze the alternative addition of UDP-glucuronic acid and UDP-N-acetylglucosamine to the nascent HA chain and extrude it through the plasma membrane. (C) Mechanism of HA degradation by HYAL enzymes and KIAA1199. In HYAL-mediated degradation of HA, HMWHA is tethered to the cell surface by CD44 and GPI-anchored HYAL2 into caveolin-rich lipid rafts and then cleaved to form 20 kDa products. The HA fragments are subsequently delivered to endo-lysosome compartments and degraded into smaller oligosaccharides by HYAL1 and βexoglycosidase enzymes. In the proposed mechanism of KIAA1199-mediated HA degradation, KIAA1199 is an HA-binding protein required for HA catabolism via the clathrin-coated pit pathway. HA depolymerization may occur to generate HA oligosaccharides while in the clathrincoated vesicles or early endosomes. Fig. 2. The HA-rich tumor microenvironment recruits and activates stromal cells to enhance tumorigenicity. (A) Monocytes and MSCs exit from the bone marrow into the circulation in cytokine- and growth factor-dependent manners. These later extravasate to target organs and become recruited into tumor stroma. HMW-HA forms a continuous meshwork with its binding molecules, which include versican, TSG-6, and inter-α-inhibitor heavy chains (HC), to cause monocyte and MSC immobilization. Upon infiltrating into tumor stroma, LMW-HA stimulates the activation and differentiation of monocytes toward an M2 phenotype. (B) Accumulation of HA in the tumor stroma drives the differentiation and activation of TAFs. Elevated levels of HA 28

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promote directional infiltration of resident tissue fibroblasts and differentiation of MSCs into TAFs under the influence of growth factors. Increased HA may drive trans-differentiation of cancer cells into TAFs. (C) HA is an essential component required for tumor angiogenesis. During angiogenesis, both HMW- and LMW-HA stimulate endothelial cell migration, proliferation, and differentiation in a manner coordinated with growth factors, such as VEGF and b-FGF. LMW-HA also drives angiogenic responses by disrupting the endothelial barrier, which induces vascular the hyperpermeability that occurs in tumor angiogenesis. Fig. 3. HA-mediated induction of EMT and cancer stemness. (A) HA can trigger cancer cells to acquire a multipotent stem cell-like phenotype through EMT induction. HA regulates the expression of EMT-related transcription factors, such as Snail and Twist, which drive CSC selfrenewal and maintenance. (B) HA is a key player in tumor metastasis by inducing EMT and promoting ECM degradation. HA/CD44 interactions promote the PI3K/Akt signaling pathway and induce the MMP-2 and MMP-9 expression involved in the invasion and metastasis of cancer cells. Following the activation of CD44, ERM proteins crosslink CD44 to the actin cytoskeleton, thereby leading to an increase in cell migration. (C) HA/CD44 interactions regulate cancer stemness and multidrug resistance. HA/CD44 linking promotes the activation of stem cell regulators Rex1 and Sox2 through Nanog. Phosphorylation of Nanog induces multidrug resistance by upregulating MDR1expression.

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