Báo cáo khoa học: Hyaluronan–CD44 interactions as potential targets for cancer therapy

M I N I R E V I E W

Hyaluronan–CD44 interactions as potential targets for cancer therapy Suniti Misra1, Paraskevi Heldin2, Vincent C. Hascall3, Nikos K. Karamanos4, Spyros S. Skandalis2, Roger R. Markwald1 and Shibnath Ghatak1

1 Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA 2 Ludwig Institute for Cancer Research, Uppsala University Biomedical Centre, Sweden 3 Department of Biomedical Engineering ⁄ ND20, Cleveland Clinic, Cleveland, OH, USA 4 Department of Chemistry, Laboratory of Biochemistry, University of Patras, Greece

Keywords cancer; CD44-varient; gene-therapy; hyaluronan; nanoparticles; stem cells; shRNA-therapy; tumorigenesis; tumor- stroma; wound-healing

studies. The downregulation of

Correspondence S. Misra, or S. Ghatak, Regenerative Medicine and Cell Biology, BSB # 613, Medical University of South Carolina, Charleston, SC 29425, USA Fax: 843 792 0664 Tel: 843 792 8642 E-mail: misra@musc.edu; ghatak@musc.edu P. Heldin, Ludwig Institute for Cancer Research, Uppsala University Biomedical Centre, Box 595, SE-75124 Uppsala, Sweden Tel: 0046 18 160414 Fax: 0046 18 160420

(Received 19 October 2010, revised 18 January 2011, accepted 25 February 2011)

doi:10.1111/j.1742-4658.2011.08071.x

It is becoming increasingly clear that signals generated in tumor microenvi- ronments are crucial to tumor cell behavior, such as survival, progression and metastasis. The establishment of these malignant behaviors requires that tumor cells acquire novel adhesion and migration properties to detach from their original sites and to localize to distant organs. CD44, an adhe- sion ⁄ homing molecule, is a major receptor for the glycosaminoglycan hyal- uronan, which is one of the major components of the tumor extracellular matrix. CD44, a multistructural and multifunctional molecule, detects changes in extracellular matrix components, and thus is well positioned to provide appropriate responses to changes in the microenvironment, i.e. engagement in cell–cell and cell–extracellular matrix interactions, cell traf- ficking, lymph node homing and the presentation of growth factors ⁄ cyto- kines ⁄ chemokines to co-ordinate signaling events that enable the cell responses that change in the tissue environment. The potential involvement of CD44 variants (CD44v), especially CD44v4–v7 and CD44v6–v9, in tumor progression has been confirmed for many tumor types in numerous clinical the standard CD44 isoform (CD44s) in colon cancer is postulated to result in increased tumorigenicity. CD44v-specific functions could be caused by their higher binding affinity than CD44s for hyaluronan. Alternatively, CD44v-specific functions could be caused by differences in associating molecules, which may bind selec- tively to the CD44v exon. This minireview summarizes how the interaction between hyaluronan and CD44v can serve as a potential target for cancer therapy, in particular how silencing CD44v can target multiple metastatic tumors.

Introduction

Ten years ago, Hanahan and Weinberg [1] proposed seven hallmarks of cancer shared by most tumor cells,

namely self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless

Abbreviations CD44s, standard CD44; CD44v, variant CD44; ECM, extracellular matrix; HA, hyaluronan; HAS, hyaluronan synthase; HGF, hepatocyte growth factor; HYAL, hyaluronidase; MMP, matrix metalloproteinase; MSCs, mesenchymal stem cells; PEG, polyethylene glycol; PEI, polyethyleneimine; PI3K, phosphoinositide 3-kinase; shRNA, short hairpin RNA; Tf, transferrin.

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sustained angiogenesis,

in which HA affects

conditions

matrix remodeling occurring during embryonic mor- phogenesis, inflammation and tumorigenesis [8–10]. HA is found in pericellular matrices attached to HA-synthe- sizing enzymes or its receptors, and is also present in intracellular compartments [11–14]. The regulation of transient interactions of HA with its HA-binding pro- teins, hyaladherins (both extracellular and cell surface receptors), is crucial for fundamental physiological pro- cesses, e.g. embryonic development, but also for patho- logical cell proliferation, migration and differentiation [10,15,16].

tissue replicative potential, invasion and metastasis. More recently, Kroemer and Pouyssegur [2] further extended these essential hall- marks of cancer with altered tumor cell-intrinsic metabolism, proposing the avoidance of immunosur- veillance as a result of metabolic reprogramming of tumor cells as another hallmark of cancer. In addition, it is now widely recognized that the tumor-associated tumor progression stroma contributes to malignant [1,3]. The tumor microenvironment contains many dis- including vascular cells, fibroblasts, tinct cell types, immune cells and components of the extracellular i.e. growth factors and cytokines, as matrix (ECM), well as structural molecules [4,5]. Tumor cells sense paracrine signals from the local microenvironment and communicate these signals with their stromal cells. In this way, they often alter the cellular and molecular composition of a particular tumor microenvironment to promote and maintain tumor progression. Hence, the notion of the tumor microenvironment as an inte- grated and essential part of the metastatic phenotype of carcinoma cells has been the subject of intense investigation. The disruption of ECM promotes abnor- mal inter- and ⁄ or intracellular signaling, leading to the dysregulation of cell proliferation, growth and cyto- skeleton reorganization [6,7].

The glycosaminoglycan hyaluronan (HA) is a major component in the ECM of most mammalian tissues, which accumulates in sites of cell division and rapid

The adhesion ⁄ homing molecule CD44, which is implicated in cell–cell and cell–matrix adhesion, is the major cell surface receptor for HA. CD44 proteins exist in three states with respect to HA binding: non- binding; nonbinding unless activated by physiological stimuli; and constitutively binding [17–19]. HA induces signaling when it binds to constitutively activated CD44 variants (CD44v) [20,21]. CD44 can also react with other molecules, including collagen, fibronectin, osteopontin, growth factors and matrix metallopro- the functional roles of such teinases (MMPs), but interactions are less well known [22]. CD44 is a trans- membrane protein encoded by a single gene, but, as a result of alternate splicing, multiple forms of CD44 are generated that are further modified by N- and O-linked glycosylations (Fig. 1). The smallest CD44 isoform that lacks variant exons, designated standard CD44 (CD44s), is abundantly expressed by both normal and

Fig. 1. Structure, binding domains and interactions of CD44. The ectodomain of CD44 contains hyaluronan-binding motifs and is decorated with chondroitin ⁄ heparan sulfate that both affect its hyaluronan-binding capacity and enable its interactions with growth factors ⁄ growth fac- tor receptors and matrix metalloproteinases (MMPs). Transmembrane and cytoplasmic domains undergo multiple post-translational modifica- tions, including palmitoylation and phosphorylation on cysteine and serine residues, respectively, promoting the binding of proteins with crucial functions in cytoskeletal organization and signaling. ErbB2, epidermal growth factor receptor-2; ERM, ezrin–radixin–moesin; FGF, fibro- blast growth factor; HGF, hepatocyte growth factor; IQGAP1, IQ motif containing GTPase activating protein 1; MAPK, mitogen-activated pro- tein kinase; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; TGFR, transforming growth factor receptor; VEGF, vascular endothelial growth factor.

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HA and CD44 in tumor initiation and progression

external

Influence of HA in tumorigenesis

Reactive stroma in cancer is often characterized by an increase in cancer-associated fibroblasts ⁄ myofibroblasts that produce an array of growth factors and chemokin- es, and amplify the synthesis of HA and proteoglycans such as versican. The interaction of the anti-adhesion molecule versican with HA and CD44 promotes the expansion of the pericellular matrix. These complexes the pericellular increase the viscoelastic nature of matrix, creating a highly malleable extracellular envi- ronment that supports the cell shape change necessary for cancer cell proliferation and migration. Further- more, versican, via its chondroitin ⁄ dermatan sulfate side-chains, is highly polyanionic, which amplifies the hydration of the environment caused by HA [54]. A large number of studies performed during the last three decades have demonstrated a close correlation between malignancy and HA-rich ECM, as well as with CD44s and CD44v expression. CD44 in cancer cells interacts with HA-rich microenvironments, which affects cell sig- naling pathways that trigger the ability of malignant cells to migrate, to invade basement membranes ⁄ ECM and to lodge at distant sites of the tumor [14,22,23,55– 58] (see also the interesting series of reviews on the Web Science of Hyaluronan Today at http://www. glycoforum.gr.jp). However, the underlying molecular mechanism whereby HA–CD44 cooperation influences the malignant phenotype and contributes to tumor progression is not yet clear.

Divergent mechanisms control

cancer cells, whereas the CD44v isoforms that contain a variable number of exon insertions (v1–v10) at the region are proximal plasma membrane expressed mostly by cancer cells. In addition, the CD44 ectodomain can be decorated with chondroitin sulfate and ⁄ or heparan sulfate enabling CD44 to bind growth factors, including fibroblast growth factor, vas- cular endothelial growth factor or hepatocyte growth factor (HGF) [22,23]. The rather short cytoplasmic tail of CD44 binds to ankyrin and ezrin–radixin–moesin proteins, providing a link to the cytoskeleton, as well as to merlin, which abrogates this binding. However, the multiple cellular functions of CD44 rely on its association with partner proteins that regulate cell migration, growth, survival and differentiation. CD44 is endogenously expressed at low levels on various cell types of normal tissues [24,25], but requires activation before binding to HA [17,18,26–29]. The CD44 struc- ture of normal cells is distinct from that of cancer cells because pathological conditions promote alternate splicing and post-translational modifications to pro- duce diversified CD44 molecules with enhanced HA binding which lead to increased tumorigenicity [30–36]. Glycosylation is required for spliced variant formation of CD44, which has high affinity to bind HA on cer- tain cell types, whereas glycosylations rich in sialic acid decrease HA binding [37–39]. For example, circulating lymphocytes express CD44, but do not bind HA until CD44 is deglycosylated on lymphocyte activation [37,40,41] and, to internalize CD44, it must be acylated [42]. This diversification of CD44v functions allows the production of specific targeting agents that will be use- ful for both diagnosis and therapy. Systemic applica- the variant 6 tion of antibodies directed against epitope and the expression of antisense CD44v6 retard colon tumor growth and metastasis in vivo [43,44]. The overexpression of the variant, high-molecular-mass iso- forms CD44v4–v7 and CD44v6–v9 in various cancers [45–52], as well as the downregulation of CD44s in colon cancer, are postulated to result in increased tumorigenicity [53], emphasizing the potential impor- tance of CD44 splice variants in cancer.

the expression of hyaluronan synthase (HAS) genes in response to stim- uli, and each HAS synthesizes HA molecules of differ- ent size and amount in a cell-type and context-specific manner. The study of HAS2-knockout mice [9,59] clearly demonstrated that HA deposition in the ECM was required for embryonic heart valve morphogenesis. In HAS2-null embryos, the endocardial cushion cells failed to undergo epithelial-to-mesenchymal transition and did not migrate to the cardiac jelly. This is partly a result of the lack of HA–CD44-induced Ras signal- ing. Importantly, this phenotype was seen only for the HAS2 isoform, indicating functional differences among the three HASs. The HA-synthesizing capacity of HASs and, specifically, HAS2 can be regulated by dimerization and ubiquitination [60]. In this study, the mutation of the HAS2 lysine residue 190, which is one major acceptor site for ubiquitin, led to total inactiva- tion of its enzymatic activity. The different roles of the three HAS isoforms are also likely to be related to

In this article, we review the tumorigenic actions of HA and its receptor CD44 that occur extensively in several malignant conditions. We also discuss potential therapeutic interventions for the development of tar- geted therapies based on an understanding of the com- munication between HA and cell surface CD44. In particular, we highlight possible roles in HA–CD44v- induced tumor growth and invasion, together with fresh insights into the enigmatic nature of CD44 splice variants, and how the suppression of HA–CD44v interactions may be a therapeutic target.

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anisms regulating tumor–stroma interplay and stromal targeting therapy. It should also be mentioned that there is a connection between HA catabolism and energy generation, most probably allowing HA to function as an alternative energy source to glucose for malignant cells [75]. Such metabolic reprogramming of tumor cells could add a further dimension to the importance of HA in cancer progression.

Function of CD44 in tumor initiation and metastatic behavior

different expression patterns [61]. Studies of nonmalig- nant cells overexpressing different HASs revealed that the high levels of HA induced by HAS3 were inversely correlated with cell motility and CD44 expression [62]. Importantly, the overproduction of HA in cancer cells, such as fibrosarcomas, breast cancer, mesotheliomas and prostate cancer, transfected with HAS1, HAS2 and HAS3 genes, triggered intracellular signaling path- ways that promoted anchorage-independent growth and invasiveness, which correlated with increased expression of CD44 [63–66]. HAS1 and its splice vari- ants were detected in multiple myeloma patients, but not in healthy donors, and were associated with poor survival of the patients [67]. Invasive and ⁄ or metastatic breast cancer cells deprived of HAS2 lost their aggres- sive phenotype [68]. These and many other studies, not referred to here because of space limitations, suggest that HA contributes in several ways to the hallmark properties of malignancy, especially anchorage-inde- pendent growth and invasiveness.

The increased deposition of HA in tumors is not a pas- sive process during malignancy; rather, it triggers sig- naling events and promotes the association between CD44 and other cell surface receptors that become acti- vated or inhibited either directly or indirectly through HA-activated CD44 [14,16,57,76]. Early studies by us and other laboratories revealed that aggressive breast carcinomas expressed high levels of CD44s and CD44v, as well as increased synthesis of HA [77,78]. More recent studies have highlighted the importance of CD44 molecules in the onset of malignant transformation. There is now increasing evidence that a small popula- tion of tumor cells (less than 0.1%), referred to as can- cer stem cells or cancer-initiating cells, exhibit stem cell properties, i.e. are responsible for maintaining the tumor and, possibly, for the formation of new tumors at metastatic loci. CD44 has been identified as an important marker of such a population of cancer stem cells in breast, pancreas and colorectal cancers [79–81]. Together, these findings suggest that CD44 plays an important role in the initiation and ⁄ or maintenance of cancer stem cells in some tumors.

Specifically, CD44s

suggesting that

the

Although many studies have shown the importance of HASs in tumor growth and malignant progression, other studies have suggested a more complex role of HA. For example, HAS2 overexpression was found to suppress the tumorigenesis of glioma cells lacking hyal- uronidase (HYAL) activity [69], and HYAL1 expres- sion promoted the HAS-mediated growth suppression and metastatic ability of prostate cancer cells [70]. Notably, the overexpression of HAS2 in colon carcino- mas that possessed HYAL1 activity promoted, whereas the overexpression of HYAL1 suppressed, tumorigene- sis in an experimental model of colon carcinoma [71]. In addition, the HA content in tissues was well corre- lated with the tumor growth rate. Additional observa- tions support the notion that HYAL1 can have both tumor-promoting and tumor-suppressing functions [72]. It is possible that excess HA synthesis and degra- dation in concert promote the metastatic phenotype of certain tumor types. However, the HA content in clini- cal samples is not always statistically correlated with tumor grade, transformation- induced HA overproduction may be a result of differ- ential upregulation of HAS isoforms and ⁄ or HYALs at different stages of malignant transformation. Recent work utilizing the mouse mammary tumor virus-Neu transgenic model conditionally expressing HAS2 high- lighted the role of HA in the promotion of the malig- nant phenotype. The growth rate of mammary tumors increased and an HA-rich intratumoral stroma was formed, which most probably established interactions between tumor and stromal cells that promoted angio- genesis and lymphangiogenesis [73,74]. This and other mouse models will be useful to further study the mech-

interacts with growth factor receptors, such as epidermal growth factor receptor-2 and platelet-derived growth factor receptor. Most importantly, the binding of HA to CD44s either stimu- lates [82,83] or inhibits [84] tyrosine phosphorylation by the associated tyrosine kinase receptors. Most prob- ably, the binding of HA to CD44 causes clustering, which triggers differential downstream events depen- dent on cell type and tissue context. Such clustering appears to be important for the trapping of MMP9 and the subsequent activation of transforming growth factor-b, which affects oncogenic functions including invasion and angiogenesis [85]. Moreover, the cluster- ing of CD44 also occurs on extensive N- and O-gly- cosylations of the variant ectodomain of CD44 that can affect the binding of HA to CD44 [22,23]. How- ever, there are also indications of clustering-indepen- dent signaling via CD44. Thus, HA dodecasaccharides, which most probably are unable to induce CD44

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clustering, induce chemokine CXCL1 secretion, result- ing in endothelial cell sprouting in a CD44-dependent manner [86]. During tumor progression, HAS and HYAL activities give rise to HA molecules of high or low molecular mass, with the capacity to bind differen- tially to CD44 and thereby modulate its function. This complexity may explain why CD44 expression is not correlated with tumor aggressiveness in neuroblasto- mas and prostate cancer [23].

As the detailed description of

Fig. 2. Proposed model for the cross-talk between tumor cells (epi- thelial cells) and tumor-associated stromal myofibroblasts. Cancer cells and stroma-derived fibroblasts influence each other’s develop- ment. The extracellular domain of CD44 variants, which contains the sequence encoded for variants of CD44 and their interaction with HA, is required for the stromal factor-dependent activation of receptor tyrosine kinases (RTK, such as hepatocyte growth fac- tor ⁄ Met) and its downstream anti-apoptotic signaling involving phosphoinositide 3-kinase (PI3K) ⁄ AKT and mitogen-activated pro- tein kinase (MAPK) pathways. Tumor-associated stromal myofibro- blast-derived hyaluronan, synthesized in response to stromal factors (such as hepatocyte growth factor) and cancer cell-derived CD44 variant, and RTK are involved in tumorigenesis.

CD44 and HA in tumors: wounds that do not heal

that

cells

types,

including

endothelial

invasiveness

an array of

cells. These

the expression of CD44v isoforms from less malignant to more advanced stages is beyond the scope of this minireview, we high- light the relevance of CD44v isoforms in cancer which seem to be suitable targets for anti-cancer therapy. In several primary and cancer cells, CD44v6 forms a ter- nary complex with HGF and its receptor c-Met. Most probably, CD44v6 presents HGF to its receptor, trig- gering receptor kinase activity and signaling pathways involving the binding of ezrin to ezrin–radixin–moesin proteins, and thus actin cytoskeleton binding and Ras activation. HGF elicits metastatic behavior in various types of cells, mostly in a paracrine fashion. In a recent study, we found that insulin-like growth fac- tor 1, transforming growth factor, prostaglandin E2 and tumor necrosis factor-a, secreted by prostate can- cer cells, stimulated the synthesis of HGF by myofi- broblasts. HGF, in turn, stimulated the production of splice variant 9 of CD44. The interaction of stromal- derived HA with the upregulated CD44v9 initiated sig- stabilized androgen receptor naling pathways functions and induced anti-apoptotic signaling [87]. Colon cancer cells exhibit the same mechanism, but utilize CD44v6 (S. Misra et al., unpublished results). Silencing the appropriate CD44v inhibits tumor cell adhesion to the tumor cell matrix and in vitro tumor cell invasion [87]. Cross-talk between the increased HA synthesized by the stromal cells, which interacts with colon tumor cell CD44v6, sustains HA–CD44v6–phos- phoinositide 3-kinase (PI3K) signaling through a posi- tive feedback loop between CD44v6 and PI3K that induces invasiveness. In addition, we have demon- strated that stromal-derived HGF stimulates the syn- thesis of metalloproteinase (MT1MMP), which induces shedding of CD44v, and promotes colon ⁄ prostate can- (S. Misra et al., unpublished cer cell results; depicted in the model in Fig. 2). Thus, thera- peutic approaches using HA–CD44v interaction with CD44v short hairpin RNA (shRNA) can target tumors at one or more of these levels: the microenvironment (stromal factors such as HGF and its inducers); recep- tor-based signals (select CD44v, Met ⁄ RTK); and signal transducers, such as PI3K ⁄ AKT or mitogen-activated protein kinase (Fig. 2).

The tumor microenvironment contains many distinct cell and their precursors, pericytes, smooth muscle cells, fibroblasts, carcinoma-associated fibroblasts, myofibroblasts, neu- trophils ⁄ eosinophils ⁄ basophils ⁄ mast cells, T ⁄ B lympho- cytes, natural killer cells and antigen-presenting cells, such as macrophages and dendritic cells [4]. The micro- environment of a solid tumor closely resembles the environment of wound healing and tissue repair sites of an injured tissue. On tissue injury, platelets are acti- vated. These activated platelets release vasoactive mediators for vascular permeability, serum fibrinogen formation and growth factors ⁄ cyto- for fibrin clot kines ⁄ matricellular proteins to initiate granulation tis- sue formation, activate fibroblasts, and induce and activate MMPs necessary for ECM remodeling. Epi- thelial and stromal cell types engage in a reciprocal sig- naling cross-talk to assist healing. The reciprocal signaling collapses after the wound is healed. In the case of tumorigenesis, the invasive inflammatory tumor cytokines ⁄ chemokines cells produce that are mitogenic for granulocytes ⁄ monocytes ⁄ macro- factors phages ⁄ fibroblasts ⁄ endothelial

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sites of

co-implantation models combining tumor cells and MSCs [102,103,108] hold great promise for therapeutic strategies [106], in which the interaction between tumor and stroma can be manipulated and studied (the concept of using MSCs for tumor therapy is depicted in the model in Fig. 3).

Therapeutic strategy involving perturbation of HA–CD44 interactions

Importance of targeting CD44v in vivo

produce

CD44v interaction with HA is known for its role in the metastatic cascade, as this interaction regulates the ability of malignant cells to activate receptor tyro- sine kinases, and to stimulate migration, invasion of basement membranes ⁄ ECM and migration to distant sites [22,57,109–115]. HA induces intracellular signal- ing when it binds to constitutively activated CD44v during cell dynamic processes, but does not do so under conditions of adult tissue homeostasis, which generally involves CD44s. The CD44 structure on normal cells is distinct from that on cancer cells because, under various physiological and pathological conditions, the local environmental pressure (stromal factors) influences alternate splicing and post-transla- tional modification to produce diversified CD44 molecules [35,36]. This diversification allows the pro- duction of specific targeting agents that will be useful for both diagnosis and therapy. Pathological condi- tions that stimulate alternate splicing and post-transla- tional modifications diversified CD44 molecules with enhanced HA binding that leads to increased tumorigenicity [30–36]. The systemic applica- tion of antibodies directed against a CD44v epitope [43] reduced the metastasizing activity of a pancreatic adenocarcinoma. The overexpression of variant, high- molecular-mass isoforms CD44v4–v7 and CD44v6–v9 in various cancers [45–52], as well as the downregula- tion of the CD44s isoform in colon cancer, has been postulated to result in increased tumorigenicity [53], emphasizing the potential importance of CD44 splice variants in cancer.

Inhibition of HA–CD44 interactions

compete with the

To explore the mechanism of constitutive HA–CD44 interactions and the consequent outcomes in cancer cells, four different methods were used. The first method uses small HA oligosaccharides ((cid:2)2.5 kDa) endogenous HA polymer that [83,96,110,116–118]. The second method overexpresses soluble HA-binding proteins (e.g. soluble CD44) that

(cytokines ⁄ chemokines) potentiate tumor growth, stim- ulate angiogenesis, induce fibroblast migration and enable metastatic spread. During this process, non- hematopoietic mesenchymal stem cells (MSCs) origi- nating from bone marrow localize to the sites of hematopoiesis, inflammation and sites of injury, as well as to solid tumors [88–90]. Inactivated MSCs have been shown to inhibit tumor growth by inhibiting a PI3K ⁄ AKT pathway in an E-cadherin- dependent manner, prompting the use of these cells as tumor inhibitory cells in vivo [91], whereas activated MSCs within the solid tumors are the source of carci- noma-associated fibroblasts that contribute to tumor growth in several ways [92,93]. Tissue injury and inflammation are accompanied by increased production of stromal HA, which, in addition to cell–cell and cell– matrix adhesion [94,95], and cell proliferation and sur- [10,83,87,96], helps to create highly hydrated vival ECM that may facilitate local cellular trafficking [97,98]. In the bone marrow, HA is also abundantly produced by both stromal and hematopoietic cells. CD44, in addition to its function to regulate cell prolif- eration ⁄ differentiation ⁄ survival ⁄ migration into tissues, is implicated in hematopoietic progenitor trafficking to the bone marrow and spleen [99–101]. The concept of the use of MSCs as delivery vehicles originates from the fact that tumors, similar to wounds, produce chemo- attractants, such as cytokines ⁄ chemokines (e.g. vascular endothelial growth factor, transforming growth factor- b), to recruit MSCs to form the supporting stroma of the tumor, and also pericytes for angiogenesis. MSCs transfected with the interferon-b gene can increase the production of interferon-b at the local site [102,103]. Likewise, Herrera et al. [104] presented a convincing case indicating that interactions between CD44 and HA influence the homing of exogenous MSCs that localize to the kidneys during acute renal failure, i.e. CD44 on exogenous cells is important in helping MSCs to local- ize to the damaged renal tissue in vivo. However, this in vivo function of MSCs depends partly on signals from the target tissue microenvironment, i.e. endothe- lial progenitor cells were used as gene delivery vehicles to the site of angiogenesis rather than to the quiescent vasculature [105]. On the basis of these observations, it is possible to deliver immune-activating cytokines ⁄ secreted proteins to the site of tumors through MSCs [103]. As human MSCs can be easily expanded in vitro and retain an extensive multipotent capacity for differ- entiation [106,107], in a recent study, we found that genetically engineered human MSCs which secrete solu- ble CD44v that acts as an antagonist to HA–CD44v signaling inhibit malignant properties in cancer cells (S. Misra et al., unpublished results). These studies and

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Fig. 3. Bone marrow-derived nonhematopoietic human mesenchymal stem cells (hMSCs) are pluripotent cells that are capable of differenti- ating into various tissue lineages, including osteoblasts, adipocytes, chondrocytes, myoblasts, hepatocytes and possibly even neural cells [107]. After systemic injection, hMSCs can selectively migrate to solid tumors, where they proliferate and become cancer-associated stromal myofibroblasts [103]. As hMSCs can be easily expanded in vitro and possess an extensive multipotent capacity for differentiation, they have been explored as vehicles for tissue repair and gene therapy [106], when they are appropriately engineered for therapy. We established that tissue-specific floxed plasmid ⁄ nanoparticle delivery is efficient for the activation of a gene of interest [120]. In a pilot study (S. Misra et al., unpublished results) using genetically modified hMSCs in nanoparticles, the tropism was altered, because the secreted proteins from trans- duced hMSCs interacted with stromal hyaluronan, and thus inhibited the malignant properties of cancer cells by more than 20-fold by per- turbing hyaluronan–CD44v interaction.

Strategies that target CD44 to perturb HA–CD44 interactions in tumors [121]

the HA–CD44v interaction at

[110,112],

solid

but

not

on

on

induction of

innate

act as competitive decoys for CD44 and thus bind to endogenous HA [83,96,110,116,117]. The third method blocks the HA–CD44 interaction specifically by treat- ing the cells with a blocking antibody against the HA-binding site of CD44 [96,104,117,119]. The fourth method inhibits the post-transcriptional expression of CD44v with CD44 siRNAs [83,87,110,112,119,120]. Although these methods yield valuable information on how epithelial cell-derived HA and its interaction with CD44v can influence malignant properties in vitro, they do not address the tumor cell responses to cell-specific perturbation of the genetic level in vitro and in vivo. In addition, by using the CD44 siRNA to interrupt HA–CD44v6 signaling processes at a cellular level it has been observed that the phenotypic changes induced by siR- NAs only persist for 1 week because of a lack of trans- fer of siRNA or the dilution of siRNA concentration after each cell division, or a lack of stability of siRNA, which limits their use in the inhibition of tumor pro- gression in vivo. Moreover, the dose of siRNA remains immune undefined, and the responses is another obstacle that will obscure the use of siRNAs as therapeutics.

HA-conjugated drugs CD44 can internalize HA [122]. Thus, HA-carrying drugs alone or encapsulated drugs in liposomes have the potential to be used as targeted drugs, as well as drug transport vehicles. Chemical groups of HA, such as the carboxylate on glucuronic acid, the N-acetylglu- cosamine hydroxyl and the reducing end, can poten- tially be used to conjugate a drug [123]. HA–drug conjugates are internalized via CD44, and the drug is released and activated mainly by intracellular enzy- matic hydrolysis [124–126]. Activated CD44 is overex- pressed their tumors, nontumorigenic counterparts. Several preclinical stud- ies have shown that HA chemically conjugated to cyto- toxic agents improves the anticancer properties of the agent in vitro [125,127,128]. Drugs with low solubility can be successfully applied when conjugated with HA. For instance, the antimitotic chemotherapeutic agent paclitaxel has low water solubility. On conjugation to HA, its solubility and CD44-dependent cellular uptake increase in vitro [126].

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HA-conjugated nanocarriers

(PEI)

complexes

HA, when conjugated to a nanocarrier, acts as a pro- tective structural component and a targeting coating. The circulation time and biodistribution (pharmacoki- netic properties) are influenced by incorporation of the targeting and cell-specific uptake properties of HA onto large carriers. Cargo liposomes or nanoparticles delivered to CD44-overexpressing cells include anti- cancer drugs (epirubicin [127], doxorubicin [129–139], paclitaxel [125,126] and mitomycin c [127,133]) as well as siRNA [140,141]. Results from the above studies using HA-targeted nanocarriers do not differ from many of the studies performed with HA–drug conju- gates.

Targeting with anti-CD44 antibodies

Anti-CD44 antibodies against highly expressed vari- ants can actively target drugs to CD44, inhibit and dis- rupt CD44–matrix interactions, occupy CD44 and induce CD44 signaling, which can cause apoptosis [142]. Anti-CD44 antibodies targeting ligands for either radiolabels or anti-cancer chemotherapeutics partially stabilize some patients [143,144]. CD44v6 is expressed in breast, cervical and colon cancers, and in squamous cell carcinomas. Thus CD44v was chosen as a model for therapy. A Phase 1 clinical trial was performed with an immunotoxin (humanized antibody coupled with a cytotoxic drug mertansine) against CD44v6 in 30 patients with incurable squamous cell carcinoma [76].Three patients showed a partial response and it was thought that the trial was successful. Unfortu- nately, one of the patients died, and the trial was abruptly withdrawn.

less, there has been considerable interest in developing nonviral vectors for gene therapy. In this regard, non- viral vectors, such as positively charged polyethylenei- mine shielded with polyethylene glycol (PEG), can be used safely to avoid the nonspe- cific interactions with nontarget cells and blood com- ponents [147]. Nonviral vectors were once limited because of their low gene transfer efficiency. However, the incorporation of various ligands, such as peptides, growth factors and proteins, or antibodies for targets highly expressed on cancer cells, has circumvented this obstacle [148]. In addition, enhanced permeability caused by the aberrant vasculature in solid tumors, and retention (known as the enhanced permeability and retention effect) of ligand-coated vectors around the receptors of tumor cells, can increase the chances for a high probability of interaction with the cells [120]. Thus, nonviral vectors can acquire high gene transfer efficiency [120]. This concept has been tested by preparing nonviral vector nanoparticles with plas- mids packed inside an outer PEG–PEI layer coated with transferrin (Tf), an iron-transporting protein [120,148], which binds with Tf receptors (Tf-R) with high affinity. Tf-R is present at much higher levels on tumor cells [120] than on phenotypically normal epi- thelial cells. The association of Tf with nanoparticles significantly enhances the transfection efficiency of shRNA generator plasmids by promoting the internali- zation of nanoparticles in dividing and nondividing [148]. through receptor-mediated endocytosis cells Finally, the uptake of nanoparticles carrying multi- ple functional domains (surface-shielding particles Tf– PEG–PEI, shRNA generator plasmids, tissue-specific promoter-driven Cre recombinase and conditionally silenced plasmid) can overcome the intracellular barri- ers for the successful delivery of the shRNA gene.

Tissue-specific deletion of CD44v signalling

The technique of using shRNA in an expression vector is an alternative strategy to stably suppress selected gene expression, which suggests that the use of shRNA expression vectors holds potential promise for thera- peutic approaches for silencing disease-causing genes [145]. There are two ways to deliver shRNA in cancer cells: using either a viral vector or a nonviral vector. Viral vectors have been used to achieve this proof of principle in animal models and, in selected cases, in human clinical trials [146]. Systemic targeting by viral vectors to the desired tissue is difficult because the host immune responses activate viral clearance. Systemic administration of a large amount of adenovirus (e.g. into the liver) can be a serious health hazard, which even caused the death of one patient [146]. Neverthe-

The newly developed cell-specific shRNA delivery approach by Misra et al. [120] confirmed that the tar- geting of the HA–CD44v6-induced signaling pathway inhibited distant colon tumor growth in Apc Min ⁄ + mice. Tissue-specific shRNA delivery was made possi- ble by the use of Cre recombinase produced in response to a colon tissue-specific promoter, which deletes the interruption between the U6 promoter and the CD44v6shRNA oligonucleotide. This approach, depicted in the model in Fig. 4, has successfully dem- onstrated that CD44v6shRNA is localized to the colon tumor cells by an endpoint assay of CD44v6 expres- sion, and by perturbation of HA–CD44v6 interaction, as reflected in the reduction in the number of tumors [120]. In our recent in vivo studies with C57Bl ⁄ 6 mice, we are optimistic that the systemic delivery of a mixture of two plasmids in Tf ⁄ nanoparticles (pARR2-

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Fig. 4. Model for delivery of short hairpin RNA (shRNA). This illustration depicts the cellular uptake of plasmid transferrin–polyethylene gly- col–polyethyleneimine (Tf–PEG–PEI) nanoparticles and the mechanism of action of shRNA. First, a pSico vector containing a U6 promoter- loxP-CMV-GFP-STOP signal-loxP-CD44vshRNA (gene of interest) is made. Second, an expression vector containing the Cre recombinase gene controlled by the tissue-specific promoter is created. Third, the two vectors are packaged in Tf-coated PEG–PEI nanoparticles that bind with Tf receptors (Tf-R) present at high levels in the targeted tumor cells. The delivery of the vectors in normal and malignant cells from the targeted tissue results in the deletion of the STOP signal and the transcription of Cre recombinase driven by the tissue-specific promoter. The target gene (CD44vshRNA) is then unlocked and transcribed through the strong U6 promoter for high expression. The normal tissue cells are not affected because they do not make the targeted CD44 variant.

plasmid

holds

CD44vshRNA-expressing potential promise for therapeutic approaches for silencing HA– CD44v signaling, and hence the downstream signaling that promotes disease-causing genes [145] (Fig. 4).

(a)

for the following reasons:

Advantages of the tumor-specific delivery of CD44vshRNA versus other therapeutic strategies

cancers

accumulate

thereby reducing potential

probasin-Cre ⁄ nanoparticles and floxed pSico-CD44v 9shRNA ⁄ nanoparticles) will target both localized and (S. Ghatak et al., metastatic prostate cancer cells unpublished results). This novel approach opens up new ways to combat cancer, and to understand tumor- igenesis in vivo, the cell-specific release of shRNA by the application of a tissue-specific promoter-driven Cre-lox mechanism; (b) silencing of the expression of the selected CD44v in target tissue cancer cells; (c) no effect on normal target tissue cells, which do not express targeted CD44v and rely on the CD44s form, which is not affected by the plasmids; (d) the target CD44vshRNA is not expressed in other types of cells because the tissue-specific pro- moter only unlocks the Cre recombinase in the tar- geted tissue cells, side- effects [120]; (e) the nanoparticles that carry plasmids are biodegradable and cleared from the system; (f) it addresses the pathophysiological role of HA–CD44v interactions in cancer; (g) it can establish diagnostic markers for the targeted cancer, including CD44v, sol- uble CD44 and HA; and (h) it can establish CD44v– HA interactions as an innovative novel therapeutic tar- get against cancer progression. Thus, the conditional suppression of gene expression by the use of a

First, this technique avoids the multiple chemical steps needed to prepare HA-conjugated cytotoxic drugs and conjugation to nanocarriers. Second, it abolishes CD44v in cancer cells only. Third, a number of cell types in normal tissues that express CD44 are not affected because they are not activated. Fourth, inflam- activated mation-associated immune cells having upregulated Tf receptors and CD44v. However, they may take up the nanoparticles, but no deletion of CD44v will take place because the promoter is not lymphocyte specific (S. Misra et al., unpublished results). To target activated lymphocytes, specific promoter-driven Cre plasmids should be used. Fifth, the accumulation of antibody in nontumor areas is a major limitation of anti-CD44 antibody therapy. Experiments so far have not produced any such effect in shRNA delivery.

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Concluding remarks

disease: novel roles for proteoglycans in malignancy and their pharmacological targeting. FEBS J 277, 3904– 3923.

7 Murphy G & Nagase H (2010) Localising matrix metal- loproteinase activities in the pericellular environment. FEBS J 278, 2–15. 8 Toole BP (2001) Hyaluronan in morphogenesis. Semin Cell Dev Biol 12, 79–87.

9 Camenisch TD, Spicer AP, Brehm-Gibson T, Biester- feldt J, Augustine ML, Calabro A Jr, Kubalak S, Klewer SE & McDonald JA (2000) Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transforma- tion of epithelium to mesenchyme. J Clin Invest 106, 349–360. 10 Toole BP (2004) Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 4, 528–539.

Despite the increasing number of studies conducted so far, a complete understanding of HA–CD44-induced signaling still remains elusive. However, both HA and CD44 appear to be vitally important from embryogene- sis to morphogenesis, in inflammation and in cancer, which accompanies the overexpression of CD44 and its splice variants and the aberrant synthesis ⁄ turnover of HA. On the basis of the above-mentioned functions of HA and its interaction with CD44, it seems likely that the impact of HA–CD44 and its variant-induced tumor growth is multifactorial. Importantly, CD44v-induced proteolysis [24,149] of the matrix facilitates the detach- ment of malignant tumor cells from their confined tumor area, and therefore promotes the spread of malig- nant tumor cells to distant sites. Moreover, partial deg- radation of HA molecules promotes angiogenesis, a vital requirement for tumor growth. Furthermore, by providing increased tissue hydration, HA molecules provide a suitable environment to support malignant cell migration, similar to cardiac cushion cell movement [9,59,150–153]. In summary, CD44 and, more specifi- cally, CD44v are promising target molecules for therapy and diagnosis, at least in some tumors.

11 Evanko SP, Parks WT & Wight TN (2004) Intracellu- lar hyaluronan in arterial smooth muscle cells: associa- tion with microtubules, RHAMM, and the mitotic spindle. J Histochem Cytochem 52, 1525–1535. 12 Hascall VC, Majors AK, De La Motte CA, Evanko SP, Wang A, Drazba JA, Strong SA & Wight TN (2004) Intracellular hyaluronan: a new frontier for inflammation? Biochim Biophys Acta 1673, 3–12.

13 Heldin P & Pertoft H (1993) Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells. Exp Cell Res 208, 422–429.

Acknowledgements

the National

14 Toole BP (2009) Hyaluronan–CD44 interactions in

cancer: paradoxes and possibilities. Clin Cancer Res 15, 7462–7468. 15 Laurent TC & Fraser JRE (1992) Hyaluronan. FASEB J 6, 2397–2404. 16 Naor D, Wallach-Dayan SB, Zahalka MA &

This work was supported, as a whole or in part, of Health Grants Institutes by P20RR021949 (to SG) and P20RR016434 (to SM, SG and RRM), HL RO1 33756 and 1 P30AR050953 (to VCH). This work was also supported by Mitral-07 CVD 04 (to RRM), Medical University of South Project Carolina University Research Council 2204000-24330 (to SM) and 2204000-24329 (to SG).

Sionov RV (2008) Involvement of CD44, a molecule with a thousand faces, in cancer dissemination. Semin Cancer Biol 18, 260–267. 17 Lesley J, Hascall VC, Tammi M & Hyman R (2000)

References

Hyaluronan binding by cell surface CD44. J Biol Chem 275, 26967–26975. 1 Hanahan D & Weinberg RA (2000) The hallmarks of cancer. Cell 100, 57–70. 18 Lesley J & Hyman R (1992) CD44 can be activated to function as an hyaluronic acid receptor in normal mur- ine T cells. Eur J Immunol 22, 2719–2723.

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS

1438

19 Lesley J, English N, Charles C & Hyman R (2000) The role of the CD44 cytoplasmic and transmembrane domains in constitutive and inducible hyaluronan bind- ing. Eur J Immunol 30, 245–253. 20 He Q, Lesley J, Hyman R, Ishihara K & Kincade PW 2 Kroemer G & Pouyssegur J (2008) Tumor cell metabo- lism: cancer’s Achilles’ heel. Cancer Cell 13, 472–482. 3 Wang F, Hansen RK, Radisky D, Yoneda T, Barcel- los-Hoff MH, Petersen OW, Turley EA & Bissell MJ (2002) Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional con- texts. J Natl Cancer Inst 94, 1494–1503. 4 Coussens LM & Werb Z (2002) Inflammation and can- cer. Nature 420, 860–867. 5 Coussens LM & Werb Z (2001) Inflammatory cells and cancer. Think different! J Exp Med 193, F23–F26. 6 Theocharis AD, Skandalis SS, Tzanakakis GN & (1992) Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition. J Cell Biol 119, 1711–1719. 21 Sleeman J, Rudy W, Hofmann M, Moll J, Herrlich P & Ponta H (1996) Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity. J Cell Biol 135, 1139–1150. Karamanos NK (2010) Proteoglycans in health and

S. Misra et al.

Targeting CD44 variants in tumors

22 Ponta H, Sherman L & Herrlich PA (2003) CD44:

from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol 4, 33–45. 38 Skelton TP, Zeng C, Nocks A & Stamenkovic I (1998) Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hy- aluronan. J Cell Biol 140, 431–446. 39 Rochman M, Moll J, Herrlich P, Wallach SB, Ned- 23 Orian-Rousseau V (2010) CD44, a therapeutic target for metastasising tumours. Eur J Cancer 46, 1271–1277. 24 Cichy J & Pure E (2003) The liberation of CD44. J Cell Biol 161, 839–843.

vetzki S, Sionov RV, Golan I, Ish-Shalom D & Naor D (2000) The CD44 receptor of lymphoma cells: struc- ture–function relationships and mechanism of activa- tion. Cell Adhes Commun 7, 331–347. 40 Hathcock KS, Hirano H, Murakami S & Hodes RJ 25 Mackay CR, Terpe HJ, Stauder R, Marston WL, Stark H & Gunthert U (1994) Expression and modulation of CD44 variant isoforms in humans. J Cell Biol 124, 71– 82.

(1993) CD44 expression on activated B cells. Differen- tial capacity for CD44-dependent binding to hyaluronic acid. J Immunol 151, 6712–6722. 26 Sneath RJ & Mangham DC (1998) The normal struc- ture and function of CD44 and its role in neoplasia. Mol Pathol 51, 191–200.

41 Lesley J, English N, Perschl A, Gregoroff J & Hyman R (1995) Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. J Exp Med 182, 431–437. 27 Lesley J, He Q, Miyake K, Hamann A, Hyman R & Kincade PW (1992) Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody. J Exp Med 175, 257–266. 28 Lesley J, Howes N, Perschl A & Hyman R (1994)

42 Thankamony SP & Knudson W (2006) Acylation of CD44 and its association with lipid rafts are required for receptor and hyaluronan endocytosis. J Biol Chem 281, 34601–34609. 43 Seiter S, Arch R, Reber S, Komitowski D, Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response. J Exp Med 180, 383–387. 29 Lesley J & Hyman R (1998) CD44 structure and func- tion. Front Biosci 3, D616–D630.

Hofmann M, Ponta H, Herrlich P, Matzku S & Zoller M (1993) Prevention of tumor metastasis formation by anti-variant CD44. J Exp Med 177, 443–455.

30 Bourguignon LY, Zhu H, Shao L, Zhu D & Chen YW (1999) Rho-kinase (ROK) promotes CD44v(3,8-10)– ankyrin interaction and tumor cell migration in meta- static breast cancer cells. Cell Motil Cytoskeleton 43, 269–287.

44 Reeder JA, Gotley DC, Walsh MD, Fawcett J & Antalis TM (1998) Expression of antisense CD44 variant 6 inhibits colorectal tumor metastasis and tumor growth in a wound environment. Cancer Res 58, 3719–3726. 45 Gunthert U, Hofmann M, Rudy W, Reber S, Zoller M,

31 Bourguignon LY, Zhu H, Shao L & Chen YW (2000) CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migra- tion. J Biol Chem 275, 1829–1838.

Haussmann I, Matzku S, Wenzel A, Ponta H & Herrlich P (1991) A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65, 13–24. 46 Naor D, Sionov RV, Zahalka M, Rochman M,

32 Bourguignon LY, Singleton PA, Zhu H & Zhou B (2002) Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells. J Biol Chem 277, 39703–39712. 33 Bourguignon LY, Zhu H, Shao L & Chen YW (2001) Holzmann B & Ish-Shalom D (1998) Organ-specific requirements for cell adhesion molecules during lymphoma cell dissemination. Curr Top Microbiol Immunol 231, 143–166.

CD44 interaction with c-Src kinase promotes cortactin- mediated cytoskeleton function and hyaluronic acid- dependent ovarian tumor cell migration. J Biol Chem 276, 7327–7336. 34 Pure E & Cuff CA (2001) A crucial role for CD44 in 47 Ochiai S, Nakanishi Y, Mizuno K, Hashimoto S, Inutsuka S, Kawasaki M, Yatsunami J & Hara N (1997) [Expression of CD44 standard and CD44 variant 6 in human lung cancer]. Nihon Kyobu Shikkan Gakkai Zasshi 35, 1179–1185. inflammation. Trends Mol Med 7, 213–221.

35 van Weering DH, Baas PD & Bos JL (1993) A PCR- based method for the analysis of human CD44 splice products. PCR Methods Appl 3, 100–106. 36 Naor D, Nedvetzki S, Golan I, Melnik L & 48 Kurozumi K, Nishida T, Nakao K, Nakahara M & Tsujimoto M (1998) Expression of CD44 variant 6 and lymphatic invasion: importance to lymph node metastasis in gastric cancer. World J Surg 22, 853–857; discussion 857–858. Faitelson Y (2002) CD44 in cancer. Crit Rev Clin Lab Sci 39, 527–579.

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS

1439

37 Katoh S, Zheng Z, Oritani K, Shimozato T & Kincade PW (1995) Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J Exp Med 182, 419– 429. 49 Foekens JA, Dall P, Klijn JG, Skroch-Angel P, Claas- sen CJ, Look MP, Ponta H, Van Putten WL, Herrlich P & Henzen-Logmans SC (1999) Prognostic value of CD44 variant expression in primary breast cancer. Int J Cancer 84, 209–215.

S. Misra et al.

Targeting CD44 variants in tumors

64 Itano N, Sawai T, Miyaishi O & Kimata K (1999)

50 Ayhan A, Tok EC & Bildirici I (2001) Overexpression of CD44 variant 6 in human endometrial cancer and its prognostic significance. Gynecol Oncol 80, 355–358. 51 Ishida T (2000) Immunohistochemical expression of Relationship between hyaluronan production and met- astatic potential of mouse mammary carcinoma cells. Cancer Res 59, 2499–2504. 65 Li Y & Heldin P (2001) Hyaluronan production the CD44 variant 6 in colorectal adenocarcinoma. Surg Today 30, 28–32. 52 Ishibashi M, Nishida T, Murakami H, Shiraishi M, increases the malignant properties of mesothelioma cells. Br J Cancer 85, 600–607. 66 Liu N, Gao F, Han Z, Xu X, Underhill CB &

Aritomi T & Yoshida M (1995) The role of interstitial hyaluronan in acute lung injury. Nihon Kyobu Shikkan Gakkai Zasshi 33(Suppl), 225–230. Zhang L (2001) Hyaluronan synthase 3 overexpression promotes the growth of TSU prostate cancer cells. Cancer Res 61, 5207–5214. 67 Adamia S, Maxwell CA & Pilarski LM (2005) Hyal-

53 Choi SH, Takahashi K, Eto H, Yoon SS & Tanabe KK (2000) CD44s expression in human colon carcinomas influences growth of liver metastases. Int J Cancer 85, 523–526. uronan and hyaluronan synthases: potential therapeutic targets in cancer. Curr Drug Targets Cardiovasc Hae- matol Disord 5, 3–14. 68 Li Y, Li L, Brown TJ & Heldin P (2007) Silencing of

hyaluronan synthase 2 suppresses the malignant pheno- type of invasive breast cancer cells. Int J Cancer 120, 2557–2567.

54 Skandalis SS, Kletsas D, Kyriakopoulou D, Stavropo- ulos M & Theocharis DA (2006) The greatly increased amounts of accumulated versican and decorin with specific post-translational modifications may be closely associated with the malignant phenotype of pancreatic cancer. Biochim Biophys Acta 1760, 1217–1225. 55 Cheng C, Yaffe MB & Sharp PA (2006) A positive

feedback loop couples Ras activation and CD44 alter- native splicing. Genes Dev 20, 1715–1720. 69 Enegd B, King JA, Stylli S, Paradiso L, Kaye AH & Novak U (2002) Overexpression of hyaluronan syn- thase-2 reduces the tumorigenic potential of glioma cells lacking hyaluronidase activity. Neurosurgery 50, 1311–1318.

70 Bharadwaj AG, Kovar JL, Loughman E, Elowsky C, Oakley GG & Simpson MA (2009) Spontaneous metastasis of prostate cancer is promoted by excess hyaluronan synthesis and processing. Am J Pathol 174, 1027–1036. 71 Jacobson A, Rahmanian M, Rubin K & Heldin P 56 Anttila MA, Tammi RH, Tammi MI, Syrjanen KJ, Saarikoski SV & Kosma VM (2000) High levels of stromal hyaluronan predict poor disease outcome in epithelial ovarian cancer. Cancer Res 60, 150–155. 57 Marhaba R & Zoller M (2004) CD44 in cancer pro- gression: adhesion, migration and growth regulation. J Mol Histol 35, 211–231.

(2002) Expression of hyaluronan synthase 2 or hyal- uronidase 1 differentially affect the growth rate of transplantable colon carcinoma cell tumors. Int J Can- cer 102, 212–219. 72 Lokeshwar VB, Cerwinka WH, Isoyama T & 58 Theocharis AD, Vynios DH, Papageorgakopoulou N, Skandalis SS & Theocharis DA (2003) Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma. Int J Biochem Cell Biol 35, 376–390.

Lokeshwar BL (2005) HYAL1 hyaluronidase in prostate cancer: a tumor promoter and suppressor. Cancer Res 65, 7782–7789. 73 Koyama H, Hibi T, Isogai Z, Yoneda M, Fujimori 59 Camenisch TD, Schroeder JA, Bradley J, Klewer SE & McDonald JA (2002) Heart-valve mesenchyme forma- tion is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors. Nat Med 8, 850–855. 60 Karousou E, Kamiryo M, Skandalis SS, Ruusala A,

Asteriou T, Passi A, Yamashita H, Hellman U, Heldin CH & Heldin P (2010) The activity of hyaluronan syn- thase 2 is regulated by dimerization and ubiquitination. J Biol Chem 285, 23647–23654. M, Amano J, Kawakubo M, Kannagi R, Kimata K, Taniguchi S et al. (2007) Hyperproduction of hyaluro- nan in neu-induced mammary tumor accelerates angiogenesis through stromal cell recruitment: possible involvement of versican ⁄ PG-M. Am J Pathol 170, 1086–1099. 74 Koyama H, Kobayashi N, Harada M, Takeoka M,

Kawai Y, Sano K, Fujimori M, Amano J, Ohhashi T, Kannagi R et al. (2008) Significance of tumor-associ- ated stroma in promotion of intratumoral lymphangio- genesis: pivotal role of a hyaluronan-rich tumor microenvironment. Am J Pathol 172, 179–193. 61 Jacobson A, Brinck J, Briskin MJ, Spicer AP & Heldin P (2000) Expression of human hyaluronan synthases in response to external stimuli. Biochem J 348(Pt 1), 29–35. 62 Brinck J & Heldin P (1999) Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44. Exp Cell Res 252, 342–351.

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS

1440

75 Smedsrod B & Seljelid R (1991) Fate of intravenously injected aminated beta(1–3) polyglucose derivatized with 125I-tyraminyl cellobiose. Immunopharmacology 21, 149–158. 63 Kosaki R, Watanabe K & Yamaguchi Y (1999) Over- production of hyaluronan by expression of the hyal- uronan synthase Has2 enhances anchorage-independent growth and tumorigenicity. Cancer Res 59, 1141–1145.

S. Misra et al.

Targeting CD44 variants in tumors

update on clinical trials with mesenchymal stem cells. J Cell Physiol 211, 27–35. 90 Sordi V, Malosio ML, Marchesi F, Mercalli A, Melzi R, 76 Orian-Rousseau V & Ponta H (2008) Adhesion pro- teins meet receptors: a common theme? Adv Cancer Res 101, 63–92. 77 Heldin P, de la Torre M, Ytterberg D & Bergh J

Giordano T, Belmonte N, Ferrari G, Leone BE, Bertuzzi F et al. (2005) Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 106, 419–427.

(1996) Differential synthesis and binding of hyaluronan by human breast cancer cell lines: relationship to hor- mone receptor status. Oncology Rep 3, 1011–1016. 78 Kaufmann M, Heider KH, Sinn HP, von Minckwitz G, Ponta H & Herrlich P (1995) CD44 variant exon epitopes in primary breast cancer and length of sur- vival. Lancet 345, 615–619.

91 Khakoo AY, Pati S, Anderson SA, Reid W, Elshal MF, Rovira II, Nguyen AT, Malide D, Combs CA, Hall G et al. (2006) Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 203, 1235–1247.

79 Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ & Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100, 3983–3988. 80 Simeone DM (2008) Pancreatic cancer stem cells: 92 Guo X, Oshima H, Kitmura T, Taketo MM & Oshima M (2008) Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer. J Biol Chem 283, 19864–19871. 93 Haniffa MA, Wang XN, Holtick U, Rae M, Isaacs implications for the treatment of pancreatic cancer. Clin Cancer Res 14, 5646–5648.

JD, Dickinson AM, Hilkens CM & Collin MP (2007) Adult human fibroblasts are potent immunoregulatory cells and functionally equivalent to mesenchymal stem cells. J Immunol 179, 1595–1604. 81 Du L, Wang H, He L, Zhang J, Ni B, Wang X, Jin H, Cahuzac N, Mehrpour M, Lu Y et al. (2008) CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res 14, 6751–6760. 94 Fraser JR, Cahill RN & Kimpton WG (1994) Hyal-

uronic acid and cell adhesion molecules in haematolo- gy. Aust N Z J Med 24, 71.

82 Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L & Hung MC (1997) Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell acti- vation. J Biol Chem 272, 27913–27918. 83 Ghatak S, Misra S & Toole BP (2005) Hyaluronan 95 Fraser JR, Laurent TC & Laurent UB (1997) Hyaluro- nan: its nature, distribution, functions and turnover. J Intern Med 242, 27–33. 96 Ghatak S, Misra S & Toole BP (2002) Hyaluronan

constitutively regulates ErbB2 phosphorylation and sig- naling complex formation in carcinoma cells. J Biol Chem 280, 8875–8883.

oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase ⁄ Akt cell survival pathway. J Biol Chem 277, 38013–38020.

84 Li L, Heldin CH & Heldin P (2006) Inhibition of plate- let-derived growth factor-BB-induced receptor activa- tion and fibroblast migration by hyaluronan activation of CD44. J Biol Chem 281, 26512–26519. 85 Yu Q & Stamenkovic I (1999) Localization of matrix 97 Hallgren R, Gerdin B & Tufveson G (1990) Hyaluronic acid accumulation and redistribution in rejecting rat kidney graft. Relationship to the transplantation edema. J Exp Med 171, 2063–2076. 98 Tufveson G, Gerdin B, Larsson E, Laurent T, metalloproteinase 9 to the cell surface provides a mech- anism for CD44-mediated tumor invasion. Genes Dev 13, 35–48.

86 Takahashi Y, Li L, Kamiryo M, Asteriou T, Mousta- kas A, Yamashita H & Heldin P (2005) Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1 ⁄ GRO1-dependent manner. J Biol Chem 280, 24195–24204. 87 Ghatak S, Hascall VC, Markwald RR & Misra S

Wallander J, Wells A & Hallgren R (1992) Hyaluronic acid accumulation; the mechanism behind graft rejection edema. Transpl Int 5(Suppl 1), S688–S689. 99 Avigdor A, Goichberg P, Shivtiel S, Dar A, Peled A, Samira S, Kollet O, Hershkoviz R, Alon R, Hardan I et al. (2004) CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem ⁄ progenitor cells to bone marrow. Blood 103, 2981–2989. 100 Khaldoyanidi S, Denzel A & Zoller M (1996) Require- ment for CD44 in proliferation and homing of hemato- poietic precursor cells. J Leukoc Biol 60, 579–592. 101 Vermeulen M, Le Pesteur F, Gagnerault MC, Mary JY, (2010) Stromal hyaluronan interaction with epithelial CD44 variants promotes prostate cancer invasiveness by augmenting expression and function of hepatocyte growth factor and androgen receptor. J Biol Chem 285, 19821–19832.

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS

1441

Sainteny F & Lepault F (1998) Role of adhesion mole- cules in the homing and mobilization of murine hemato- poietic stem and progenitor cells. Blood 92, 894–900. 88 Mendes SC, Robin C & Dzierzak E (2005) Mesenchy- mal progenitor cells localize within hematopoietic sites throughout ontogeny. Development 132, 1127–1136. 102 Nakamizo A, Marini F, Amano T, Khan A, Studeny M, Gumin J, Chen J, Hentschel S, Vecil G, 89 Giordano A, Galderisi U & Marino IR (2007) From the laboratory bench to the patient’s bedside: an

S. Misra et al.

Targeting CD44 variants in tumors

squamous cell carcinomas of the head and neck. J Pathol 179, 66–73. 115 Regidor PA, Callies R, Regidor M, Gunthert U, Dembinski J et al. (2005) Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65, 3307–3318.

Zoller M & Schindler AE (1996) Expression of the CD44 variant isoforms 6 and 4 ⁄ 5 in breast cancer. Correlation with established prognostic parameters. Arch Gynecol Obstet 258, 125–135.

103 Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE & Andreeff M (2004) Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 96, 1593–1603. 104 Herrera MB, Bussolati B, Bruno S, Morando L, 116 Misra S, Ghatak S, Zoltan-Jones A & Toole BP (2003) Regulation of multidrug resistance in cancer cells by hyaluronan. J Biol Chem 278, 25285–25288. 117 Zoltan-Jones A, Huang L, Ghatak S & Toole BP

Mauriello-Romanazzi G, Sanavio F, Stamenkovic I, Biancone L & Camussi G (2007) Exogenous mesenchy- mal stem cells localize to the kidney by means of CD44 following acute tubular injury. Kidney Int 72, 430–441. (2003) Elevated hyaluronan production induces mesen- chymal and transformed properties in epithelial cells. J Biol Chem 278, 45801–45810. 105 Anderson SA, Glod J, Arbab AS, Noel M, Ashari P,

118 Marieb EA, Zoltan-Jones A, Li R, Misra S, Ghatak S, Cao J, Zucker S & Toole BP (2004) Emmprin pro- motes anchorage-independent growth in human mam- mary carcinoma cells by stimulating hyaluronan production. Cancer Res 64, 1229–1232. 119 Zhu H, Mitsuhashi N, Klein A, Barsky LW, Fine HA & Frank JA (2005) Noninvasive MR imaging of magnetically labeled stem cells to directly identify neovasculature in a glioma model. Blood 105, 420–425. 106 Gafni Y, Turgeman G, Liebergal M, Pelled G, Gazit Z & Gazit D (2004) Stem cells as vehicles for orthopedic gene therapy. Gene Ther 11, 417–426. 107 Gregory CA, Prockop DJ & Spees JL (2005) Non-

Weinberg K, Barr ML, Demetriou A & Wu GD (2006) The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells 24, 928–935. hematopoietic bone marrow stem cells: molecular con- trol of expansion and differentiation. Exp Cell Res 306, 330–335.

120 Misra S, Hascall VC, De Giovanni C, Markwald RR & Ghatak S (2009) Delivery of CD44 shRNA ⁄ nano- particles within cancer cells: perturbation of hyaluro- nan ⁄ CD44v6 interactions and reduction in adenoma growth in Apc Min ⁄ + mice. J Biol Chem 284, 12432–12446. 108 Kanehira M, Xin H, Hoshino K, Maemondo M, Miz- uguchi H, Hayakawa T, Matsumoto K, Nakamura T, Nukiwa T & Saijo Y (2007) Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mes- enchymal stem cells. Cancer Gene Ther 14, 894–903. 109 Marhaba R, Bourouba M & Zoller M (2005) CD44v6

promotes proliferation by persisting activation of MAP kinases. Cell Signal 17, 961–973. 121 Platt VM & Szoka FC Jr (2008) Anticancer therapeu- tics: targeting macromolecules and nanocarriers to hyaluronan or CD44, a hyaluronan receptor. Mol Pharm 5, 474–486. 122 Culty M, Nguyen HA & Underhill CB (1992) The

hyaluronan receptor (CD44) participates in the uptake and degradation of hyaluronan. J Cell Biol 116, 1055– 1062. 110 Misra S, Ghatak S & Toole BP (2005) [JBC Paper of the Week]) Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyal- uronan, phosphoinositide 3-kinase, and ErbB2. J Biol Chem 280, 20310–20315.

123 Pouyani T & Prestwich GD (1994) Functionalized deriv- atives of hyaluronic acid oligosaccharides: drug carriers and novel biomaterials. Bioconjug Chem 5, 339–347. 124 Luo Y, Kirker KR & Prestwich GD (2000) Cross-

111 Misra S, Obeid LM, Hannun YA, Minamisawa S, Berger FG, Markwald RR, Toole BP & Ghatak S (2008) Hyaluronan constitutively regulates activation of COX-2-mediated cell survival activity in intestinal epithelial and colon carcinoma cells. J Biol Chem 283, 14335–14344. linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. J Control Release 69, 169–184. 112 Misra S, Toole BP & Ghatak S (2006) Hyaluronan

125 Luo Y & Prestwich GD (1999) Synthesis and selective cytotoxicity of a hyaluronic acid–antitumor bioconju- gate. Bioconjug Chem 10, 755–763. constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells. J Biol Chem 281, 34936–34941. 113 Heldin P, Karousou E, Bernert B, Porsch H,

126 Luo Y, Ziebell MR & Prestwich GD (2000) A hyal- uronic acid–taxol antitumor bioconjugate targeted to cancer cells. Biomacromolecules 1, 208–218. 127 Akima K, Ito H, Iwata Y, Matsuo K, Watari N, Nishitsuka K & Skandalis SS (2008) Importance of hyaluronan–CD44 interactions in inflammation and tumorigenesis. Connect Tissue Res 49, 215–218. 114 Herold-Mende C, Seiter S, Born AI, Patzelt E, Schupp

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS

1442

Yanagi M, Hagi H, Oshima K, Yagita A, Atomi Y et al. (1996) Evaluation of antitumor activities of hyaluronate binding antitumor drugs: synthesis, M, Zoller J, Bosch FX & Zoller M (1996) Expression of CD44 splice variants in squamous epithelia and

S. Misra et al.

Targeting CD44 variants in tumors

characterization and antitumor activity. J Drug Target 4, 1–8. degradable hyaluronic acid nanogels. J Control Release 119, 245–252. 128 Coradini D, Pellizzaro C, Miglierini G, Daidone MG 141 Yun YH, Goetz DJ, Yellen P & Chen W (2004)

Hyaluronan microspheres for sustained gene delivery and site-specific targeting. Biomaterials 25, 147– 157. 142 Song G, Liao X, Zhou L, Wu L, Feng Y & Han ZC & Perbellini A (1999) Hyaluronic acid as drug delivery for sodium butyrate: improvement of the anti-prolifera- tive activity on a breast-cancer cell line. Int J Cancer 81, 411–416. 129 Eliaz RE & Szoka FC Jr (2001) Liposome-encapsu-

lated doxorubicin targeted to CD44: a strategy to kill CD44-overexpressing tumor cells. Cancer Res 61, 2592–2601.

130 Eliaz RE, Nir S, Marty C & Szoka FC Jr (2004) Deter- mination and modeling of kinetics of cancer cell killing by doxorubicin and doxorubicin encapsulated in tar- geted liposomes. Cancer Res 64, 711–718. 131 Eliaz RE, Nir S & Szoka FC Jr (2004) Interactions

of hyaluronan-targeted liposomes with cultured cells: modeling of binding and endocytosis. Methods Enzymol 387, 16–33. 132 Peer D, Dekel Y, Melikhov D & Margalit R (2004)

(2004) HI44a, an anti-CD44 monoclonal antibody, induces differentiation and apoptosis of human acute myeloid leukemia cells. Leuk Res 28, 1089–1096. 143 Tijink BM, Buter J, de Bree R, Giaccone G, Lang MS, Staab A, Leemans CR & van Dongen GA (2006) A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus. Clin Cancer Res 12, 6064–6072. 144 Sauter A, Kloft C, Gronau S, Bogeschdorfer F, Erhardt T, Golze W, Schroen C, Staab A, Riechelmann H & Hoermann K (2007) Pharmaco- kinetics, immunogenicity and safety of bivatuzumab mertansine, a novel CD44v6-targeting immunoconju- gate, in patients with squamous cell carcinoma of the head and neck. Int J Oncol 30, 927–935. Fluoxetine inhibits multidrug resistance extrusion pumps and enhances responses to chemotherapy in syngeneic and in human xenograft mouse tumor models. Cancer Res 64, 7562–7569. 133 Peer D & Margalit R (2004) Loading mitomycin C 145 Paul CP, Good PD, Winer I & Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20, 505–508.

inside long circulating hyaluronan targeted nano-lipo- somes increases its antitumor activity in three mice tumor models. Int J Cancer 108, 780–789. 134 Peer D & Margalit R (2000) Physicochemical evalua-

146 Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, Wilson JM & Batshaw ML (2003) Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80, 148–158. tion of a stability-driven approach to drug entrapment in regular and in surface-modified liposomes. Arch Biochem Biophys 383, 185–190.

147 Kursa M, Walker GF, Roessler V, Ogris M, Roedl W, Kircheis R & Wagner E (2003) Novel shielded transfer- rin–polyethylene glycol–polyethylenimine ⁄ DNA com- plexes for systemic tumor-targeted gene transfer. Bioconjug Chem 14, 222–231. 148 Bellocq NC, Pun SH, Jensen GS & Davis ME (2003)

Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery. Bioconjug Chem 14, 1122–1132. 135 Peer D & Margalit R (2004) Tumor-targeted hyaluro- nan nanoliposomes increase the antitumor activity of liposomal doxorubicin in syngeneic and human xeno- graft mouse tumor models. Neoplasia 6, 343–353. 136 Yadav AK, Agarwal A, Rai G, Mishra P, Jain S, Mishra AK, Agrawal H & Agrawal GP (2010) Development and characterization of hyaluronic acid decorated PLGA nanoparticles for delivery of 5-fluorouracil. Drug Deliv 17, 561–572. 149 Itoh Y & Seiki M (2006) MT1-MMP: a potent modi-

fier of pericellular microenvironment. J Cell Physiol 206, 1–8. 137 Yadav AK, Mishra P & Agrawal GP (2008) An insight on hyaluronic acid in drug targeting and drug delivery. J Drug Target 16, 91–107. 150 West DC, Hampson IN, Arnold F & Kumar S (1985) 138 Yadav AK, Mishra P, Jain S, Mishra P, Mishra AK &

Angiogenesis induced by degradation products of hyal- uronic acid. Science 228, 1324–1326. 151 West DC & Kumar S (1989) Hyaluronan and angio- genesis. Ciba Found Symp 143, 187–201.

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS

1443

152 West DC & Kumar S (1991) Tumour-associated hyal- uronan: a potential regulator of tumour angiogenesis. Int J Radiat Biol 60, 55–60. 153 Camenisch TD & McDonald JA (2000) Hyaluronan: is Agrawal GP (2008) Preparation and characterization of HA-PEG-PCL intelligent core-corona nanoparticles for delivery of doxorubicin. J Drug Target 16, 464–478. 139 Yadav AK, Mishra P, Mishra AK, Mishra P, Jain S & Agrawal GP (2007) Development and characterization of hyaluronic acid-anchored PLGA nanoparticulate carriers of doxorubicin. Nanomedicine 3, 246–257. 140 Lee H, Mok H, Lee S, Oh YK & Park TG (2007) Target-specific intracellular delivery of siRNA using bigger better? Am J Respir Cell Mol Biol 23, 431–433.