MINIREVIEW 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 Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA Ludwig Institute for Cancer Research, Uppsala University Biomedical Centre, Sweden Department of Biomedical Engineering ⁄ ND20, Cleveland Clinic, Cleveland, OH, USA Department of Chemistry, Laboratory of Biochemistry, University of Patras, Greece Keywords cancer; CD44-varient; gene-therapy; hyaluronan; nanoparticles; stem cells; shRNA-therapy; tumorigenesis; tumorstroma; wound-healing 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 microenvironments 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 adhesion ⁄ homing molecule, is a major receptor for the glycosaminoglycan hyaluronan, 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 trafficking, lymph node homing and the presentation of growth factors ⁄ cytokines ⁄ 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 studies The downregulation of 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 selectively 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 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1429 Targeting CD44 variants in tumors S Misra et al replicative potential, sustained angiogenesis, tissue invasion and metastasis More recently, Kroemer and Pouyssegur [2] further extended these essential hallmarks of cancer with altered tumor cell-intrinsic metabolism, proposing the avoidance of immunosurveillance 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 stroma contributes to malignant tumor progression [1,3] The tumor microenvironment contains many distinct cell types, including vascular cells, fibroblasts, immune cells and components of the extracellular matrix (ECM), i.e growth factors and cytokines, as 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 integrated and essential part of the metastatic phenotype of carcinoma cells has been the subject of intense investigation The disruption of ECM promotes abnormal inter- and ⁄ or intracellular signaling, leading to the dysregulation of cell proliferation, growth and cytoskeleton 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 matrix remodeling occurring during embryonic morphogenesis, inflammation and tumorigenesis [8–10] HA is found in pericellular matrices attached to HA-synthesizing enzymes or its receptors, and is also present in intracellular compartments [11–14] The regulation of transient interactions of HA with its HA-binding proteins, hyaladherins (both extracellular and cell surface receptors), is crucial for fundamental physiological processes, e.g embryonic development, but also for pathological conditions in which HA affects cell proliferation, migration and differentiation [10,15,16] 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: nonbinding; 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 metalloproteinases (MMPs), but the functional roles of such interactions are less well known [22] CD44 is a transmembrane 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 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 factor receptors and matrix metalloproteinases (MMPs) Transmembrane and cytoplasmic domains undergo multiple post-translational modifications, 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, fibroblast growth factor; HGF, hepatocyte growth factor; IQGAP1, IQ motif containing GTPase activating protein 1; MAPK, mitogen-activated protein kinase; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; TGFR, transforming growth factor receptor; VEGF, vascular endothelial growth factor 1430 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS S Misra et al cancer cells, whereas the CD44v isoforms that contain a variable number of exon insertions (v1–v10) at the proximal plasma membrane external region are 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, vascular 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 structure of normal cells is distinct from that of cancer cells because pathological conditions promote alternate splicing and post-translational modifications to produce 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 certain cell types, whereas glycosylations rich in sialic acid decrease HA binding [37–39] For example, circulating lymphocytes express CD44, but 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 useful for both diagnosis and therapy Systemic application of antibodies directed against the variant 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 isoforms 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 importance of CD44 splice variants in cancer 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 targeted therapies based on an understanding of the communication between HA and cell surface CD44 In particular, we highlight possible roles in HA–CD44vinduced 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 Targeting CD44 variants in tumors HA and CD44 in tumor initiation and progression 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 chemokines, 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 increase the viscoelastic nature of the pericellular matrix, creating a highly malleable extracellular environment that supports the cell shape change necessary for cancer cell proliferation and migration Furthermore, 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 signaling 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 the expression of hyaluronan synthase (HAS) genes in response to stimuli, and each HAS synthesizes HA molecules of different 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 signaling 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 inactivation of its enzymatic activity The different roles of the three HAS isoforms are also likely to be related to FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1431 Targeting CD44 variants in tumors S Misra et al different expression patterns [61] Studies of nonmalignant 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 pathways that promoted anchorage-independent growth and invasiveness, which correlated with increased expression of CD44 [63–66] HAS1 and its splice variants 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 aggressive 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-independent growth and invasiveness 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 hyaluronidase (HYAL) activity [69], and HYAL1 expression promoted the HAS-mediated growth suppression and metastatic ability of prostate cancer cells [70] Notably, the overexpression of HAS2 in colon carcinomas that possessed HYAL1 activity promoted, whereas the overexpression of HYAL1 suppressed, tumorigenesis in an experimental model of colon carcinoma [71] In addition, the HA content in tissues was well correlated with the tumor growth rate Additional observations support the notion that HYAL1 can have both tumor-promoting and tumor-suppressing functions [72] It is possible that excess HA synthesis and degradation in concert promote the metastatic phenotype of certain tumor types However, the HA content in clinical samples is not always statistically correlated with tumor grade, suggesting that the transformationinduced HA overproduction may be a result of differential 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 highlighted the role of HA in the promotion of the malignant 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 angiogenesis and lymphangiogenesis [73,74] This and other mouse models will be useful to further study the mech1432 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 The increased deposition of HA in tumors is not a passive process during malignancy; rather, it triggers signaling events and promotes the association between CD44 and other cell surface receptors that become activated 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 population of tumor cells (less than 0.1%), referred to as cancer 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 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 stimulates [82,83] or inhibits [84] tyrosine phosphorylation by the associated tyrosine kinase receptors Most probably, the binding of HA to CD44 causes clustering, which triggers differential downstream events dependent 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 clustering of CD44 also occurs on extensive N- and O-glycosylations of the variant ectodomain of CD44 that can affect the binding of HA to CD44 [22,23] However, there are also indications of clustering-independent signaling via CD44 Thus, HA dodecasaccharides, which most probably are unable to induce CD44 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS S Misra et al clustering, induce chemokine CXCL1 secretion, resulting 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 differentially to CD44 and thereby modulate its function This complexity may explain why CD44 expression is not correlated with tumor aggressiveness in neuroblastomas and prostate cancer [23] As the detailed description of the expression of CD44v isoforms from less malignant to more advanced stages is beyond the scope of this minireview, we highlight 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 ternary complex with HGF and its receptor c-Met Most probably, CD44v6 presents HGF to its receptor, triggering 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 factor 1, transforming growth factor, prostaglandin E2 and tumor necrosis factor-a, secreted by prostate cancer cells, stimulated the synthesis of HGF by myofibroblasts HGF, in turn, stimulated the production of splice variant of CD44 The interaction of stromalderived HA with the upregulated CD44v9 initiated signaling pathways that stabilized androgen receptor 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–phosphoinositide 3-kinase (PI3K) signaling through a positive feedback loop between CD44v6 and PI3K that induces invasiveness In addition, we have demonstrated that stromal-derived HGF stimulates the synthesis of metalloproteinase (MT1MMP), which induces shedding of CD44v, and promotes colon ⁄ prostate cancer cell invasiveness (S Misra et al., unpublished results; depicted in the model in Fig 2) Thus, therapeutic 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); receptor-based signals (select CD44v, Met ⁄ RTK); and signal transducers, such as PI3K ⁄ AKT or mitogen-activated protein kinase (Fig 2) Targeting CD44 variants in tumors Fig Proposed model for the cross-talk between tumor cells (epithelial cells) and tumor-associated stromal myofibroblasts Cancer cells and stroma-derived fibroblasts influence each other’s development 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 factor ⁄ Met) and its downstream anti-apoptotic signaling involving phosphoinositide 3-kinase (PI3K) ⁄ AKT and mitogen-activated protein kinase (MAPK) pathways Tumor-associated stromal myofibroblast-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 not heal The tumor microenvironment contains many distinct cell types, including endothelial cells and their precursors, pericytes, smooth muscle cells, fibroblasts, carcinoma-associated fibroblasts, myofibroblasts, neutrophils ⁄ eosinophils ⁄ basophils ⁄ mast cells, T ⁄ B lymphocytes, natural killer cells and antigen-presenting cells, such as macrophages and dendritic cells [4] The microenvironment of a solid tumor closely resembles the environment of wound healing and tissue repair sites of an injured tissue On tissue injury, platelets are activated These activated platelets release vasoactive mediators for vascular permeability, serum fibrinogen for fibrin clot formation and growth factors ⁄ cytokines ⁄ matricellular proteins to initiate granulation tissue formation, activate fibroblasts, and induce and activate MMPs necessary for ECM remodeling Epithelial and stromal cell types engage in a reciprocal signaling cross-talk to assist healing The reciprocal signaling collapses after the wound is healed In the case of tumorigenesis, the invasive inflammatory tumor cells produce an array of cytokines ⁄ chemokines that are mitogenic for granulocytes ⁄ monocytes ⁄ macrophages ⁄ fibroblasts ⁄ endothelial cells These factors FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1433 Targeting CD44 variants in tumors S Misra et al (cytokines ⁄ chemokines) potentiate tumor growth, stimulate angiogenesis, induce fibroblast migration and enable metastatic spread During this process, nonhematopoietic mesenchymal stem cells (MSCs) originating from bone marrow localize to the sites of hematopoiesis, sites of 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-cadherindependent 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 carcinoma-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 survival [10,83,87,96], helps to create highly hydrated 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 proliferation ⁄ 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 chemoattractants, such as cytokines ⁄ chemokines (e.g vascular endothelial growth factor, transforming growth factorb), 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 localize 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 endothelial 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 differentiation [106,107], in a recent study, we found that genetically engineered human MSCs which secrete soluble 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 1434 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 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 tyrosine kinases, and to stimulate migration, invasion of basement membranes ⁄ ECM and migration to distant sites [22,57,109–115] HA induces intracellular signaling when it binds to constitutively activated CD44v during cell dynamic processes, but does not 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-translational modification to produce diversified CD44 molecules [35,36] This diversification allows the production of specific targeting agents that will be useful for both diagnosis and therapy Pathological conditions that stimulate alternate splicing and post-translational modifications produce diversified CD44 molecules with enhanced HA binding that leads to increased tumorigenicity [30–36] The systemic application of antibodies directed against a CD44v epitope [43] reduced the metastasizing activity of a pancreatic adenocarcinoma The overexpression of variant, highmolecular-mass isoforms CD44v4–v7 and CD44v6–v9 in various cancers [45–52], as well as the downregulation 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 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 (2.5 kDa) that compete with the endogenous HA polymer [83,96,110,116–118] The second method overexpresses soluble HA-binding proteins (e.g soluble CD44) that FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS S Misra et al Targeting CD44 variants in tumors Fig Bone marrow-derived nonhematopoietic human mesenchymal stem cells (hMSCs) are pluripotent cells that are capable of differentiating 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 transduced hMSCs interacted with stromal hyaluronan, and thus inhibited the malignant properties of cancer cells by more than 20-fold by perturbing hyaluronan–CD44v interaction 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 treating 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 not address the tumor cell responses to cell-specific perturbation of the HA–CD44v interaction at 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 [110,112], it has been observed that the phenotypic changes induced by siRNAs only persist for week because of a lack of transfer 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 progression in vivo Moreover, the dose of siRNA remains undefined, and the induction of innate immune responses is another obstacle that will obscure the use of siRNAs as therapeutics Strategies that target CD44 to perturb HA–CD44 interactions in tumors [121] 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-acetylglucosamine hydroxyl and the reducing end, can potentially be used to conjugate a drug [123] HA–drug conjugates are internalized via CD44, and the drug is released and activated mainly by intracellular enzymatic hydrolysis [124–126] Activated CD44 is overexpressed on solid tumors, but not on their nontumorigenic counterparts Several preclinical studies have shown that HA chemically conjugated to cytotoxic 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] FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1435 Targeting CD44 variants in tumors S Misra et al HA-conjugated nanocarriers HA, when conjugated to a nanocarrier, acts as a protective structural component and a targeting coating The circulation time and biodistribution (pharmacokinetic 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 anticancer 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 not differ from many of the studies performed with HA–drug conjugates Targeting with anti-CD44 antibodies Anti-CD44 antibodies against highly expressed variants can actively target drugs to CD44, inhibit and disrupt 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 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 Unfortunately, one of the patients died, and the trial was abruptly withdrawn 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 therapeutic 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] Neverthe1436 less, there has been considerable interest in developing nonviral vectors for gene therapy In this regard, nonviral vectors, such as positively charged polyethyleneimine (PEI) complexes shielded with polyethylene glycol (PEG), can be used safely to avoid the nonspecific interactions with nontarget cells and blood components [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 plasmids 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 epithelial cells The association of Tf with nanoparticles significantly enhances the transfection efficiency of shRNA generator plasmids by promoting the internalization of nanoparticles in dividing and nondividing cells through receptor-mediated endocytosis [148] Finally, the uptake of nanoparticles carrying multiple 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 barriers for the successful delivery of the shRNA gene The newly developed cell-specific shRNA delivery approach by Misra et al [120] confirmed that the targeting of the HA–CD44v6-induced signaling pathway inhibited distant colon tumor growth in Apc Min ⁄ + mice Tissue-specific shRNA delivery was made possible 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 demonstrated that CD44v6shRNA is localized to the colon tumor cells by an endpoint assay of CD44v6 expression, 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 ⁄ mice, we are optimistic that the systemic delivery of a mixture of two plasmids in Tf ⁄ nanoparticles (pARR2- FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS S Misra et al Targeting CD44 variants in tumors Fig Model for delivery of short hairpin RNA (shRNA) This illustration depicts the cellular uptake of plasmid transferrin–polyethylene glycol–polyethyleneimine (Tf–PEG–PEI) nanoparticles and the mechanism of action of shRNA First, a pSico vector containing a U6 promoterloxP-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 not make the targeted CD44 variant probasin-Cre ⁄ nanoparticles and floxed pSico-CD44v 9shRNA ⁄ nanoparticles) will target both localized and metastatic prostate cancer cells (S Ghatak et al., unpublished results) This novel approach opens up new ways to combat cancer, and to understand tumorigenesis in vivo, for the following reasons: (a) 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 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 promoter only unlocks the Cre recombinase in the targeted tissue cells, thereby reducing potential sideeffects [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, soluble CD44 and HA; and (h) it can establish CD44v– HA interactions as an innovative novel therapeutic target against cancer progression Thus, the conditional suppression of gene expression by the use of a CD44vshRNA-expressing plasmid holds potential promise for therapeutic approaches for silencing HA– CD44v signaling, and hence the downstream signaling that promotes disease-causing genes [145] (Fig 4) Advantages of the tumor-specific delivery of CD44vshRNA versus other therapeutic strategies 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, inflammation-associated cancers accumulate activated 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 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1437 Targeting CD44 variants in tumors S Misra et al Concluding remarks 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 embryogenesis 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 detachment of malignant tumor cells from their confined tumor area, and therefore promotes the spread of malignant tumor cells to distant sites Moreover, partial degradation 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 specifically, CD44v are promising target molecules for therapy and diagnosis, at least in some tumors 10 11 12 13 Acknowledgements 14 This work was supported, as a whole or in part, by the National 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Am J Respir Cell Mol Biol 23, 431–433 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1443 ... highmolecular-mass isoforms CD44v4–v7 and CD44v6–v9 in various cancers [45–52], as well as the downregulation of the CD44s isoform in colon cancer, has been postulated to result in increased tumorigenicity... Ras signaling 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... 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 pathways