MINIREVIEW

MicroRNA regulation of proteoglycan function in cancer € tte2 Sherif A. Ibrahim1, Hebatallah Hassan1 and Martin Go 1 Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt €nster University Hospital, Germany 2 Department of Gynecology and Obstetrics, Mu

Keywords cancer; cancer stem cells; ceRNA; decorin; glypican; heparanase; heparan sulfate; microRNA; post-transcriptional regulation; syndecan Correspondence €tte, Department of Gynecology and M. Go €nster University Hospital, Obstetrics, Mu Albert-Schweitzer-Campus 1, D11, 48149 M€ unster, Germany Fax: +49 251 8355928 Tel: +49 251 8356117 E-mail: [email protected]

MicroRNAs are small noncoding RNAs acting as physiological regulators of gene expression at the post-transcriptional level. In cancer, the expression of microRNAs is dysregulated compared to healthy tissue, suggesting a mechanistic role in disease progression. Recent experimental evidence supports the important molecular role of proteoglycans as microRNA targets in this process. Misexpression of specific microRNAs results in aberrant expression patterns of proteoglycans, as well as their biosynthetic enzymes. Consequently, cell proliferation and apoptosis, adhesion, migration, invasiveness, epithelial-to-mesenchymal transition and cancer stem cell properties are affected as a result of the multifunctional properties of proteoglycans. A pharmacological targeting of the microRNA–proteoglycan axis emerges as a new therapeutic concept in cancer.

(Received 23 May 2014, revised 8 August 2014, accepted 26 August 2014) doi:10.1111/febs.13026

Introduction Cancer is a leading cause of mortality within the global population [1]. Therapeutic targeting is hampered by the complexity of the disease, which includes not only molecular changes within the tumor cell itself, but also within its microenvironment [2,3]. Tumor angiogenesis, tumor–stroma interactions, and interactions with immune cells, as well as with the extracellular matrix (ECM) and cancer stem cell niches, allow for malignant cell survival and the promotion of metastasis, the leading cause for cancer-associated mortality [3,4]. Proteoglycans (PGs) and glycosaminoglycans (GAGs), comprising structurally diverse constituents of the ECM and cell surfaces, have emerged as novel biomarkers and molecular players both within tumor cells and their microenvironment because they

integrate signals from growth factors, chemokines and integrins, and cell–cell as well as matrix adhesion [4,5]. Importantly, their expression is dysregulated in numerous tumor entities, with the potential to modulate all molecular events relevant to tumor progression [4–12]. Notably, altered expression of PGs and GAGs in cancer is not only regulated via classical transcription factor-mediated mechanisms, but also as a result of epigenetic modifications [12,13]. Recent studies have demonstrated that the expression of PGs and enzymes mediating the biosynthesis and degradation of their GAG carbohydrate chains is regulated by microRNAs (miRNAs), which are small noncoding RNAs capable of regulating gene expression at the post-transcriptional and translational levels [14–16]. In the present

Abbreviations ceRNA, competing endogenous RNA; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; FGF, fibroblast growth factor; GAG, glycosaminoglycan; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MAPK, mitogen-activated protein kinase; miRNA, microRNA; MMP, matrix metalloproteinase; NDST1, N-deacetylase/N-sulfotransferase-1; PG, proteoglycan; TGF, transforming growth factor.

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review, we introduce the pathophysiological role of these novel regulators and their PG targets in tumor progression and also discuss the implications of this functional interplay for the development of novel diagnostic and therapeutic options in malignant disease.

miRNAs: a novel class of posttranscriptional regulators in cancer miRNAs are a conserved class of small 19–24-nucleotide noncoding RNAs that regulate gene expression post-transcriptionally in a sequence-specific manner [14–16]. Most miRNAs are initially transcribed in the nucleus by RNA polymerase II [17] as long capped and polyadenylated primary transcripts (pri-miRNAs) that undergo processing by the endonuclease Drosha and its binding partner DGCR8/Pasha, resulting in an ~ 70nucleotide stem-loop RNA (pre-miRNA). Pre-miRNAs are exported into the cytoplasm via the nuclear pore complex constituent Exportin 5 and cleaved by the RNAse III-like enzyme Dicer, resulting in ~ 22-nucleotide RNA duplexes [18]. Following processing by helicase, the strand with decreased base-pairing at its 50 end is selected to function as a mature miRNA, whereas the complementary passenger strand (the *-miRNA strand) is degraded [15,19]. The mature miRNA associates with Argonaute and accessory proteins, thus forming the RNA-induced silencing complex. miRNAs mainly act by mRNA deadenylation and degradation through recruitment of the CCR4: NOT complex in animals [20,21]. A 6–7-nucleotide seed sequence in the 50 end of the single-stranded miRNA forms base pairs with target mRNAs involving predominantly their 30 UTRs [15,22]. Depending on the degree of sequence complementarity, the target mRNA is either degraded, or gene expression is inhibited at the translational level [14–16]. Although miRNAs are mainly negative regulators, upregulated gene expression can be mediated via binding to the 50 UTR of target genes, or via miRNA-mediated inhibition of transcriptional repressors [16,23–25]. Further regulatory complexity is achieved via transcriptional coregulation of target genes with miRNAs located in introns, via transcription of microRNA clusters, and via epigenetic regulation [25–27]. Considering that each of the over 2500 mature human miRNAs [28] can target several mRNAs, that an individual mRNA may be targeted by multiple miRNAs, and that targeting at the translational level may not require full sequence complementarity, it can be easily conceived that almost every cellular process may be regulated by miRNAs [25,29]. Indeed, individual miRNAs and miRNA signatures have a prognostic value for distinct tumor entities 5010

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[30–32] and can predict the likelihood of a successful antitumoral therapeutic intervention [33–35], distinguish different subtypes of cancer within one tumor entity [36] and distinguish different types of cancer when used as a serum marker [36]. Although these findings emphasize the clinicopathological relevance of miRNAs, experimental evidence from in vitro and in vivo models has confirmed a mechanistic contribution of altered microRNA expression to almost all steps of cancer progression [3,18,37]. Below, we describe several examples of miRNA-dependent modulation of tumor progression that are linked to a dysregulation of target PGs.

PGs: multifunctional modulators of cancer progression PGs consist of a core protein to which at least one carbohydrate chain of the glycosaminoglycan type is attached [4,7,8]. GAGs are long, unbranched polysaccharides composed of repeating disaccharide units of alternating uronic acids and amino sugars [4,7,12,13]. Four major GAG classes with relevance to cancer have been identified: the heparin-related heparan sulfate (HS), chondroitin sulfate/dermatan sulfate, keratan sulfate and the nonsulfated hyaluronan, which is not covalently linked to a core protein [7,12,13]. By contrast to nucleic acids and proteins, biosynthesis of GAGs proceeds in a nontemplate-driven process in the endoplasmic reticulum and Golgi apparatus [4]. Posttranslational modifications such as epimerization and sulfation result in enormous structural diversity and formation of specific binding motifs for numerous ligands relevant to tumor progression [4–6]. Notably, dysregulated expression of PGs and GAGs in cancer correlates with clinical prognosis in several malignant neoplasms [4,6,8,10,11]. Tumorigenesis and cancer progression are influenced by numerous dysregulated molecular and cellular events, which have been conceptually summarized as ‘hallmarks of cancer’ by Hanahan and Weinberg [3]. Remarkably, numerous studies have indicated that dysregulated PG function mechanistically contributes to each of these hallmarks [4,8,9] (Fig. 1). For example, cell surface HS PGs (HSPGs) serve as coreceptors for growth factors, thus enhancing proliferative signaling and tumor growth [4,38]. Dysregulation of small interstitial PGs such as decorin results in aberrant signaling via the growth suppressing cytokine transforming growth factor (TGF)-ß, and has an impact on tumor cell death [8,39]. Notably, the results of recent studies demonstrate an important impact of cell surface HSPGs on wnt-signaling-dependent cancer stem cell phenotypes as a prerequisite for FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

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Fig. 1. Pathogenetic involvement of PGs in the hallmarks of cancer. Dysregulated expression of PGs modulates several key events in tumor progression, the so-called hallmarks of cancer [3]. (1) As classical co-receptors for receptor tyrosine kinases, they can sustain and enhance proliferative signaling [4,6]. (2) The dermatan sulfate-PG decorin and some members of the syndecan family are capable of modulating TGFb signaling, thus influencing the process of evading growth suppression [8]. (3) Decorin, syndecan-1 and additional PGs modulate signaling pathways that lead to a resistance to cell death [4,8]. (4) The HSPG syndecan-1 has been implicated in modulating a cancer stem cell phenotype, which is linked to its role in wnt-signaling [40,41]. Enhanced stemness contributes to replicative immortality. (5) Several PG families and the HS-degrading enzyme heparanase are capable of regulating tumor angiogenesis, by sequestering angiogenic factors such as the vascular endothelial growth factor (VEGF) in the ECM, by promoting the release of active angiogenic factor-GAG complexes, and by promoting angiogenic integrin signaling [6,42–45]. (6) PGs modulate tumor cell invasion and metastasis by regulating the activity of ECMdegrading enzymes such as MMPs and heparanase [6,11,38,45], by promoting integrin-associated signaling pathways, which enhance cell motility [5,43,46], by enhancing the activity of chemokines as coreceptors [5,51,52], by influencing tumor cell-endothelial cell interactions [12,48], and by modulating the proinvasive process of EMT [5,8]. These functions are closely linked to the tumor microenvironment and to a regulatory role of PGs in inflammation [44,49,51,52] . VEGFR, VEGF receptor; FN, fibronectin.

enabling replicative immortality [7,40,41]. Tumor angiogenesis is modulated by cell surface PGs of the syndecan family [6,42,43], basement membrane PGs [44] and GAG-degrading enzymes such as heparanase [6,45] via mechanisms involving altered cytokine and integrin signaling and a modulation of matrix metalloproteinase (MMP) activity [11]. Similar PG-dependent pathways are involved in activating invasion and metastasis, which includes a modulation of cytoskeletal function and cell motility [5,6,46,47], enhancement of proteolytic activity [6,11,12,38], modulation of cancer cell-endothelial interactions [12,48] and induction of epithelial-to-mesenchymal transition (EMT) [4,5,8]. Finally, PGs in the tumor microenvironment profoundly affect tumor promoting properties in distant metastatic sites [44,49], and cancer-associated inflammation and immune responses [49,50]. Indeed, PGs can facilitate formation of chemokine gradients, influence proinflammatory signal transduction via toll-like receptors, interleukins, interferons and chemokines, FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

and modulate the interaction of leukocyte integrins and selectins with their ligands [40,50–52]. Overall, these findings mark PG as multifunctional regulators of cancer progression, and as attractive molecular targets for therapeutic approaches [4,6,11,44].

miRNA-dependent regulation of PGs in malignant disease PGs have been classified according to the amino acid sequence homology of their core proteins, their GAG substitution (HS versus chondroitin sulfate/dermatan sulfate versus keratan sulfate) or their subcellular localization, distinguishing cell surface from ECM and basement membrane PGs [4,44]. Because some PGs can be substituted with more than one type of GAG chain [4], we use the latter classification to discuss selected examples of microRNA-dependent regulation of PG expression as a mechanism of regulating cancer cell behavior. Although the chondroitin sulfate-PG, 5011

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63 Yiu GK, Kaunisto A, Chin YR & Toker A (2011) NFAT promotes carcinoma invasive migration through glypican-6. Biochem J 440, 157–166. 64 Alshalalfa M, Bader GD, Bismar TA & Alhajj R (2013) Coordinate microRNA-mediated regulation of protein complexes in prostate cancer. PLoS One 8, e84261. 65 Hannafon BN, Sebastiani P, de las Morenas A, Lu J & Rosenberg CL (2011) Expression of microRNA and their gene targets are dysregulated in preinvasive breast cancer. Breast Cancer Res 13, R24. 66 Li R, Zhang L, Jia L, Duan Y, Li Y, Wang J, Bao L & Sha N (2014) MicroRNA-143 targets Syndecan-1 to repress cell growth in melanoma. PLoS One 9, e94855. 67 Schneider C, K€assens N, Greve B, Hassan H, Sch€ uring AN, Starzinski-Powitz A, Kiesel L, Seidler DG & G€ otte M (2013) Targeting of syndecan-1 by microribonucleic acid miR-10b modulates invasiveness of endometriotic cells via dysregulation of the proteolytic milieu and interleukin-6 secretion. Fertil Steril 99, 871– 881.e1.. 68 Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, Ivarsson Y, Depoortere F, Coomans C, Vermeiren E et al. (2012) Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol 14, 677–685. 69 Kharaziha P, Ceder S, Li Q & Panaretakis T (2012) Tumor-derived exosomes: a message in a bottle. Biochim Biophys Acta 1826, 103–111. 70 Soria-Valles C, Gutierrez-Fernandez A, Guiu M, Mari B, Fueyo A, Gomis RR & L opez-Otın C (2014) The anti-metastatic activity of collagenase-2 in breast cancer cells is mediated by a signaling pathway involving decorin and miR-21. Oncogene 33, 3054– 3063. 71 Benet M, Dulman RY, Suzme R, de Miera EV, Vega ME, Nguyen T, Zavadil J & Pellicer A (2012) Wild type N-ras displays anti-malignant properties, in part by downregulating decorin. J Cell Physiol 227, 2341– 2351. 72 Lee DY, Jeyapalan Z, Fang L, Yang J, Zhang Y, Yee AY, Li M, Du WW, Shatseva T & Yang BB (2010) Expression of versican 30 -untranslated region modulates endogenous microRNA functions. PLoS One 5, e13599. 73 Fang L, Du WW, Yang X, Chen K, Ghanekar A, Levy G, Yang W, Yee AJ, Lu WY, Xuan JW et al. (2013) Versican 30 -untranslated region (30 -UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity. FASEB J 27, 907–919. 74 Yang W & Yee AJ (2014) Versican 30 -untranslated region (30 UTR) promotes dermal wound repair and fibroblast migration by regulating miRNA activity. Biochim Biophys Acta 1843, 1373–1385.

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75 Ebert MS & Sharp PA (2010) Emerging roles for natural microRNA sponges. Curr Biol 20, R858–R861. 76 Kosik KS (2013) Molecular biology: circles reshape the RNA world. Nature 495, 322–324. 77 Tay Y, Rinn J & Pandolfi PP (2014) The multilayered complexity of ceRNA crosstalk and competition. Nature 505, 344–352. 78 Denzler R, Agarwal V, Stefano J, Bartel DP & Stoffel M (2014) Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol Cell 54, 766–776. 79 Rutnam ZJ, Wight TN & Yang BB (2013) miRNAs regulate expression and function of extracellular matrix molecules. Matrix Biol 32, 74–85. 80 Hu H, Li S, Cui X, Lv X, Jiao Y, Yu F, Yao H, Song E, Chen Y, Wang M et al. (2013) The overexpression of hypomethylated miR-663 induces chemotherapy resistance in human breast cancer cells by targeting heparin sulfate proteoglycan 2 (HSPG2). J Biol Chem 288, 10973–10985. 81 Liang D, Meyer L, Chang DW, Lin J, Pu X, Ye Y, Gu J, Wu X & Lu K (2010) Genetic variants in MicroRNA biosynthesis pathways and binding sites modify ovarian cancer risk, survival, and treatment response. Cancer Res 70, 9765–9776. 82 Permuth-Wey J, Chen Z, Tsai YY, Lin HY, Chen YA, Barnholtz-Sloan J, Birrer MJ, Chanock SJ, Cramer DW, Cunningham JM et al. & Ovarian Cancer Association Consortium (OCAC). (2011) MicroRNA processing and binding site polymorphisms are not replicated in the Ovarian Cancer Association Consortium. Cancer Epidemiol Biomarkers Prev. 20:1793–1797. 83 Shi X, Su S, Long J, Mei B & Chen Y (2011) MicroRNA-191 targets N-deacetylase/Nsulfotransferase 1 and promotes cell growth in human gastric carcinoma cell line MGC803. Acta Biochim Biophys Sin (Shanghai) 43, 849–856. 84 Kasza Z, Fredlund Fuchs P, Tamm C, Eriksson AS, O’Callaghan P, Heindryckx F, Spillmann D, Larsson E, Le Jan S, Eriksson I et al. (2013) MicroRNA-24 suppression of N-deacetylase/N-sulfotransferase-1 (NDST1) reduces endothelial cell responsiveness to VEGFA. J Biol Chem 288, 25956–25963. 85 Vives RR, Seffouh A & Lortat-Jacob H (2014) Postsynthetic regulation of hs structure: the yin and yang of the sulfs in cancer. Front Oncol 3, 331. 86 Bao L, Yan Y, Xu C, Ji W, Shen S, Xu G, Zeng Y, Sun B, Qian H, Chen L et al. (2013) MicroRNA-21 suppresses PTEN and hSulf-1 expression and promotes hepatocellular carcinoma progression through AKT/ ERK pathways. Cancer Lett 337, 226–236. 87 Takei Y, Takigahira M, Mihara K, Tarumi Y & Yanagihara K (2011) The metastasis-associated microRNA miR-516a-3p is a novel therapeutic target

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gastric, ovarian and breast cancer, as well as mesothelioma, whereas it is upregulated in neuroblastoma, Wilm’s tumor, hepatocellular carcinoma and melanoma, indicating context-dependent functions in tumorigenesis [4,55]. Notably, mutations in the gene for glypican-3 have been linked to the pathogenesis of Simpson–Golabi–Behmel syndrome, an overgrowth syndrome characterized by a deregulated balance between cell proliferation and apoptosis, and also by a higher incidence of embryonic tumors [4,56]. A number of studies demonstrate that expression of glypican3 in cancer is regulated by miRNAs, with important functional implications [57–60]. Indeed, glypican-3 is differentially regulated by wide range of miRNAs. This may reflect context-dependent actions of glypicans, which can act in a stimulatory manner to stabilize the interaction of Wnts with the Frizzled signaling receptors, whereas glypican-3 inhibits hedgehog signaling by competing with its receptor patched [4,55,56]. A recent study on hepatocellular carcinoma revealed that five out of over 870 screened miRNAs could target glypican-3 [57]. Although miR-129-1-3p, miR-1291 and miR-1303 upregulated glypican-3, miR-96 and miR-1271 induced its downregulation. At the functional level, glypican-3-dependent growth inhibition and an induction of cell death by miR-1271 was demonstrated [57]. In the case of miR-1291, an indirect mode of glypican-3 regulation was revealed [58]. This miRNA represses the endoplasmic reticulum stress sensor IRE1a, which cleaves the 30 UTR of glypican-3 mRNA, resulting in an indirect upregulation of glypican-3 expression upon miR-1291 upregulation. In hepatocellular carcinoma cells, miR-520c-3p expression is negatively correlated with glypican-3 protein levels [59]. Functional analysis revealed that miR-520c-3p does indeed target glypican-3, leading to an inhibition of cell proliferation, invasion and apoptosis induction [59]. Moreover, glypican-3 targeting by miR-219-5p resulted in cell cycle arrest at the G1 to S transition and, consequently, an inhibition of hepatocellular carcinoma cell proliferation [60]. Overall, these data are in accordance with the role of glypican-3 in regulating cell proliferation and survival [4,55,56]. Apart from glypican-3, a role for glypican-1 in cancer is well documented. Being upregulated in breast and pancreatic cancer, this HSPG promotes cancer cell growth by enhancing growth factor-dependent signal transduction [4]. However, glypican-1 apparently not only promotes tumor growth as a cancer cell-autonomous factor, but also has an impact on tumor angiogenesis: Lentiviral overexpression of miR-149 suppresses tumor-induced neovascularization in vivo, as well as in vitro angiogenesis in response to FGF-2 via targeting of glypican-1 FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

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and FGFR-1 [61]. miR-149 is apparently located in an intron of the glypican-1 gene, suggesting the presence of an autoregulatory loop that fine-tunes the angiogenic response to FGF-2. Preliminary work suggests a potential miRNA-dependent regulation of additional glypicans. A study on the HEK293 cell line indicated that miR-125a controls cell proliferation by targeting glypican-4, resulting in reduced signaling via the mitogen-activated protein kinase (MAPK) pathway [62], which is abberantly activated in a number of tumor types [12]. Finally, mRNA expression of glypican-6, a HSPG promoting invasive migration of breast cancer cells by enhancing Wnt5a signaling [63], is downregulated upon transfection with miR-142-3p in MCF-7 breast cancer cells (M. G€ otte and A. Engbers, unpublished data). Besides glypicans, the syndecan family of transmembrane-anchored HSPGs has multiple important roles in cancer progression [4–6] (Figs 1 and 3). miRNA expression studies have revealed an association of defined miRNA signatures with syndecan expression. For example, miRNA-1 and miRNA-16 have been identified as master regulators of the functionally related nuclear factor-kB-, Ras- and syndecan-pathways involved in prostate cancer progression [64], whereas syndecan-1 expression is negatively correlated with miR-10b in early stages of breast cancer [65] and with miR-143 in melanoma [66]. Among the four members of the syndecan family, syndecan-1 is upregulated in breast, prostate, head and neck cancer, as well as myeloma, whereas it is downregulated in lung, endometrial and cervical cancer [4,42]. Notably, a prognostic value has been assigned to altered syndecan-1 expression in several of these cancer types, emphasizing its clinicopathological relevance [4,6,10]. Dysregulated syndecan-1 expression as a consequence of altered miRNA expression has been mechanistically linked to changes in cancer cell invasiveness, proliferation and apoptosis (Fig. 3). In the early stages of breast cancer progression, expression of miR-10b, a miRNA associated with metastasis, is inversely correlated with syndecan-1 expression [65]. This finding prompted us to investigate a potential functional link between miR-10b and syndecan-1 in breast cancer. Using a miRNA overexpression approach, we demonstrated that miR-10b downregulates syndecan-1 via interactions with its 30 UTR in human MDA-MB-231 and MCF-7 breast cancer cells [46]. Both miR-10b upregulation and syndecan-1 siRNA knockdown increased in vitro invasiveness of MDA-MB-231 cells. Notably, the expression of a syndecan-1 cDNA construct lacking its endogenous 30 UTR reverted this phenotype, confirming the contribution of this HSPG 5013

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Fig. 3. Targeting of the heparan sulfate proteoglycan syndecan-1 by miR-10b induces increased breast cancer cell motility and invasiveness. Upregulation of miR-10b in breast cancer leads to a downregulation of syndecan-1 expression [5,46,65]. Syndecan-1 depletion is associated with an increased MDA-MB-231 breast cancer cell adhesion to the ECM substrates fibronectin and laminin, possibly as a secondary consequence of increased integrin activation [5,46]. The resulting increased signaling through FAK and Rho-GTPase pathways increases resistance to radiotherapy and cell motility. Cellular invasiveness is further promoted via RUNX1dependent downregulation of E-cadherin, and ATF-2-associated upregulation of protease expression. In addition, cell proliferation is increased via differential regulation of p21WAF and CDK6. Black arrows indicate syndecan-1-dependent up- or downregulation, respectively. For details, see Hassan et al. [5] and Ibrahim et al. [46].

to the miR-10b-induced phenotype. Subsequent transcriptomic and functional analysis of syndecan-1depleted MDA-MD-231 cells revealed that increased cell motility, invasiveness and resistance to radiation therapy were mechanistically linked to increased activation of b-integrins and integrin-associated pathways [5,46]. Apart from an upregulation of members of the Rho-GTPase family of cytoskeletal regulators and increased activation of focal adhesion kinase, a downregulation of the antiinvasive cell–cell adhesion molecule E-cadherin [42] and of components of the wnt-signaling pathway were observed in these cells, consistent with the pro-invasive phenotype [5,40,46] (Fig. 3). Interestingly, in endometriosis, a benign disease characterized by locally invasive growth of ectopic endometrial tissue, miR-10b-dependent downregulation of syndecan-1 resulted in reduced invasiveness, which could be attributed to reduced MMP expression and 5014

reduced activation of the HGF/c-Met pathway [67], demonstrating that both the action of miRNAs and PGs are context-dependent. In melanoma, miR-143 expression is downregulated and shows a correlation with clinical staging [66]. Moreover, its expression is inversely correlated with syndecan-1, suggesting a potential functional link. Recent in vitro studies demonstrated that miR-143 targets the 30 UTR of syndecan-1, resulting in suppressed melanoma cell proliferation, an enhancement of G1 phase cell cycle arrest and an induction of apoptosis [66]. Overall, these results emphasize the role of miRNAs in regulating cancer cell phenotypes by targeting the multifunctional HSPGs of the syndecan family. An additional, indirect link between cell surface HSPGs and miRNA function is based on their property of acting as endocytosis receptors [54]. Previous work had demonstrated a mechanistic involvement of HSPGs of the syndecan family in the biogenesis and secretion of exosomes [68,69]. These small multivesicular bodyderived vesicles have emerged as novel means of intercellular communication, partially as a result of serving as carriers for microRNAs [69]. Recent work in glioblastoma cells has shown that exosome endocytosis and intracellular activation of exosome-mediated MAPK signaling depends on cell surface HSPGs. Internalized exosomes colocalized with syndecans and glypicans and their uptake was inhibited by exogenous HS, as well as in cells with impaired HS biosynthesis [54]. These data suggest that cell surface HSPGs play pivotal roles in the secretion and activation of exosomes, and therefore the intercellular distribution of miRNAs, with implications for communication between tumor cells and their microenvironment [69].

Extracellular matrix PGs Extracellular PGs are important ECM constituents. They contribute to the structural organization and mechanoelastical properties of the ECM, serve as a reservoir for growth factors, and participate in signaling processes relevant to cancer progression [4,8,9,44] (Fig. 1). Concerning miRNA-dependent regulation in cancer, decorin, a small leucine rich repeat PG [8] and members of the hyaluronan- and lectin-binding hyalectan PG family [4] are of particular interest. Decorin is a physiological regulator of collagen fibrillogenesis and TGF-b signaling, which is upregulated in osteosarcoma, testicular tumors, ovarian, colon, gastric, pancreatic, laryngeal, liver and breast cancer [4,8,39]. By influencing signal transduction through receptor tyrosine kinases, such as members of the epidermal growth factor receptor/ErbB family and cMet, decorin FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

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modulates tumor angiogenesis, tumor cell proliferation, adhesion and apoptosis [4,8] (Fig. 1). In a pioneering study, Merline et al. [50] demonstrated that decorin regulates signaling implicated in inflammation and tumor growth via downregulation of miR-21, a translational inhibitor of the tumor suppressor PDCD4. Mechanistically, cleavage of decorin by MMP8 was shown to cause reduced TGF-b signaling in breast cancer cells, resulting in downregulation of miR-21 and upregulation of PDCD4 [70]. As a consequence, tumor growth and lung metastasis of MDA-MB-231 in a xenograft model were reduced. Interestingly, the anti-malignant properties of wildtype N-Ras in colon and breast cancer cell models have been partially attributed to a downregulation of decorin, miR-29A and let-7b [71]; however, a functional link between these miRNAs and the PG has not yet been established. Among the large PGs of the hyalectan family, the chondroitin sulfate PG versican has emerged as a mechanistically intriguing regulator of miRNA function. Versican is a prognostic factor and is upregulated in a large number of tumor entities [4]. Dysregulation of versican has been linked to tumor cell proliferation, cell adhesion, motility, metastasis and tumor angiogenesis [4,9]. Recent studies have revealed that the 30 UTR of versican may act as an endogenous microRNA sponge, capable of sequestering miRNAs [72–74], thus neutralizing their activity. microRNA sponges have been initially used as a technical means for neutralizing miRNAs in experimental settings to derepress miRNA targets [75]. Subsequent work has shown that endogenous microRNA sponges may exist, which comprise circular RNAs [76] and competing endogenous RNAs (ceRNAs) [77]. The ceRNA hypothesis not only is supported by the identification of endogenous miRNA sponges in plants, prokaryotes and viruses, but also by the putative ceRNA activity of pseudogenes such as PTENP1 and KRAS1P [75,77]. However, the biological relevance of the ceRNA hypothesis has recently been challenged by quantitative assessment [78] and remains an active controversy in the field. If the hypothesis holds true, it will expand the regulatory capacity on cancer-related processes beyond the role of the PG. In the case of versican, upregulation of its 30 UTR resulted in decreased murine breast cancer cell proliferation and decreased tumor growth in a syngeneic in vivo model [72]. These findings were mechanistically related to interactions of miR-199a-3p and miR-144 with the versican 30 UTR, leading to an upregulation of the miRNA-targeted cell cycle regulator Rb, which was relieved from translational inhibition. Similarly, miR-144 and miR-136, two miRNAs targeting PTEN, interact with the versican FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

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ceRNA, leading to an indirect upregulation of the tumor suppressor [72]. Further work in hepatocellular carcinoma cells and in transgenic mice overexpressing the versican 30 UTR demonstrated increased tumor cell survival, migration, invasion and colony formation in vitro, increased endothelial cell proliferation, and an induction of hepatocarcinogenesis in the mouse model [73]. These findings could be attributed to interactions of the ceRNA with miRNAs miR-133a, miR-199a*, miR-144 and miR-431, which resulted in a upregulation of several miRNA targets, including tumorpromoting isoforms of versican, the cell surface adhesion protein and blood vessel marker CD34, and the interstitial matrix protein fibronectin. Utilizing fibroblasts and transgenic mice expressing the versican 30 UTR, Yang and Yee [74] demonstrated enhanced wound closure and fibroblast migration, which could be attributed to competitive binding of the ceRNA to miR-185, miR-203*, miR-690, miR-680 and miR-4343p, which target b-catenin, and to a concomitant upregulation of versican and ß-catenin expression. This regulatory loop may be also of interest in cancer, considering the role of cancer-associated fibroblasts in the tumor microenvironment, and the importance of ßcatenin for EMT. The key features of the postulated ceRNA function of versican are summarized in Fig. 4. Apart from versican, less is known about miRNA regulation of additional matrix PGS in cancer. The large hyalectan aggrecan is degraded and its expression is reduced during cartilage destruction in laryngeal squamous cell cancer [4]. Studies focussing on the process of chondrogenic differentiation and the pathogenesis of osteoarthritis have demonstrated miRNA targeting of aggrecan by miR-1, miR-146a and miR-181a [25,79]; however, the relevance for malignant disease has not yet been established.

Basement membrane PGs The basement membranes of epithelial and endothelial cell layers constitute an important hindrance to metastatic spread. Tumor cells utilize proteases and heparanase to degrade the basement membrane in order to metastasize to distant sites within the body [6,11,45] (Fig. 1). Apart from large matrix glycoproteins such as type IV collagen and laminins, the HSPG perlecan and collagen type XVIII are important constituents of basement membranes [4,44]. Perlecan is an important promoter of tumor growth, which is upregulated in liver cancer, oral tumors and melanoma [4]. Via its HS chains, perlecan binds angiogenic growth factors and promotes receptor activation, which leads to increased tumor cell proliferation and angiogenesis [4,44]. 5015

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Regarding tumor angiogenesis, perlecan can exert context-dependent effects because its C-terminal domain, endorepellin, negatively affects this process by blocking endothelial cell migration and capillary morphogenesis in an integrin-dependent manner [4]. A study on chemotherapy resistant subclones of the breast cancer cell line MDA-MB-231 revealed that activation of the miR-663 gene was increased by hypomethylation compared to parental cells [80]. Downregulation of miR-663 increased cellular chemosensitivity. Notably, perlecan was identified as a regulatory target of miR663 in this context, consistent with recent findings of a role of 3-O-sulfated heparan sulfate in the response of breast cancer cells to chemotherapy [12]. Similar to perlecan-derived endorepellin, proteolytic fragments of the basement membrane PG collagen XVIII called endostatin are potent inhibitors of tumor angiogenesis [4,44]. In accordance with this role in cancer, single nucleotide polymorphisms of a predicted miR-594 binding site in the a1 chain of collagen XVIII were initially identified as part of a single nucleotide polymorphism signature associated with increased risk for epithelial ovarian cancer in a study on over 350 cancer patients [81]. However, further investigations in an independent collective of over 3500 patients could not confirm these results [82].

Enzymes involved in GAG biosynthesis and degradation As noted earlier, PG function is largely determined by ligand interactions of the GAG chains, which depend 5016

β-catenin

Fig. 4. Versican transcripts modulate tumor cell behavior by acting as putative ceRNAs. The 30 UTR of versican is capable of binding to a large number of miRNAs, which are subsequently not so readily available for repressing additional targets. This postulated ceRNA function of versican results in an indirect upregulation of tumor-promoting isoforms of versican itself, of fibronectin, of the endothelial glycoprotein CD34 and of ß-catenin, leading to enhanced hepatocellular carcinoma growth and increased fibroblast motility and dermal wound repair in vivo. For details, see text; see also Lee et al. [72], Fang et al. [73] and Yang and Yee [74].

on specific sulfation and epimerization patterns. For example, we recently demonstrated an impact of altered 3-O-sulfation of HS on MAPK-induced expression of proteolytic factors and cadherin family members in breast cancer cells, which profoundly affected their invasive behavior [12]. Notably, expression of the HS biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (NDST1), which mediates N-deacetylation and N-sulfation of GlcNAc residues during early steps of HS biosynthesis, was shown to be downregulated by miR-191 [83], a miRNA highly expressed in gastric cancer. Targeting of NDST1 by this miRNA resulted in enhanced cell growth and resistance to apoptosis in a human gastric carcinoma cell line. Of potential relevance to tumor angiogenesis is the finding that targeting of NDST1 by miR-24 leads to reduced HS sulfation, reduced binding of the angiogenic factor vascular endothelial growth factor, and a resulting lower chemotactic responsiveness of endothelial cells [84]. Postsynthetic structural modifications of GAG chains are known to affect tumor cell behavior [6,45,85]. The secreted 6-O endosulfatases HSulf1 and HSulf2 remove 6-O sulfate residues of N-glucosamine present on highly sulfated HS, resulting in altered responses to growth factor signaling [85]. Their expression is dysregulated in a variety of tumor types, resulting in changes in tumor angiogenesis, tumor growth responses and resistance to apoptosis, whereby Sulfs can either promote or suppress these processes in a context-dependent manner [85]. In hepatocellular carcinoma, suppression of Hsulf-1 and PTEN expression by miR-21 promoted tumor progression, including FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

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EMT, cell proliferation and xenograft growth via activation of the Akt/extracellular signal regulated kinase pathway, which is in line with the role of HSulf1 in growth factor-mediated signaling [86]. Moreover, ectopic overexpression of miR-516a-3p in highly metastatic human scirrhous gastric carcinoma cells leads to a direct targeting of Hsulf-1, attenuating metastatic dissemination via downregulation of Wnt3a, Wnt5a and nuclear b-catenin [87]. Finally, the endo-b-D-glucuronidase heparanase has a profound effect on cancer metastasis, tumor angiogenesis and on the inflammatory microenvironment, via removal of steric hindrances for metastatic tumor cells in basement membranes, by affecting signaling via HS-binding angiogenic and growth factors, by modulating MMP levels, and by inducing shedding of syndecans, thus converting these molecules into paracrine effectors [4,6,45]. Of note, heparanase expression is regulated by miRNAs in cancer. An indirect upregulation of heparanase in breast cancer cells was observed upon miR10b-mediated targeting of syndecan-1 [46]. A direct regulation was observed in the case of miR-1258, which is inversely correlated with heparanase expression in nonsmall cell lung cancer and breast cancer [88–90]. Functional analysis revealed that heparanase downregulation by miR-1258 reduced lung cancer cell invasiveness in vitro [89], and invasiveness and brain metastasis of breast cancer cells in vivo [90], emphasizing the relevance of miRNA-dependent heparanase regulation for cancer metastasis.

Outlook: therapeutic and diagnostic potential of miRNA-dependent PG targeting in cancer Because miRNA-dependent targeting of PGs profoundly affects multiple steps in tumor progression (Figs 1 and 2), could such knowledge be utilized to improve cancer diagnostics or therapeutics? As separate entities, both PGs and miRNAs are used for diagnostic purposes. A prognostic or predictive value has been assigned to dysregulation of multiple PGs in numerous tumor entities [4,8–10]. Similarly, prognostic and predictive values have been determined for altered miRNA expression in cancerous tissues and blood of tumor patients, facilitated by the high biological stability of miRNAs in clinical specimens, and the possibility of highly sensitive and specific detection by quantitative PCR-based methods and next generation sequencing [18,25,30–35]. New diagnostic possibilities may arise from the future use of single nucleotide polymorphisms for miRNA binding sites in PG genes, and from a possibly enhanced diagnosFEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS

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tic value for the coevaluation of inverse correlations between the expression of a particular miRNA and its PG target. PGs and their GAG chains have emerged as novel therapeutic targets in cancer. Approaches used in preclinical models range from the the use of inhibitory peptides directed against or derived from core proteins, inhibitory antibodies, GAG-glycomimetics and heparinoids, to inhibitors of GAG biosynthetic or degrading enzymes [4,6,11,45]. Examples for such approaches include a coupling of syndecan-1 antibodies to radionuclides [91] or toxins [92], the use of antibodies targeting the HS chains of glypican-3 to inhibit Wnt signaling [93], or the use of decorin [8], or syndecan-1- [43], versican [9,44]- or collagen XVIII-derived peptides [4,44], to inhibit metastasis and tumor angiogenesis, respectively. Although these approaches have been successful in preclinical models, the use of heparanase inhibitors (partially effective against HSulf family members) has already reached the stage of clinical trials [6,45,94–97]. Inhibitors include SST0001, a nonanticoagulant N-acetylated, glycol split heparin, which shows synergistic effects with radiation therapy [94], and phosphomannopentaose sulfate (PI-88), which is evaluated in several phase I-III clinical trials on advanced melanoma, hepatocellular carcinoma, nonsmall cell lung cancer and prostate cancer, and which has reached phase III clinical development for hepatocellular carcinoma [6,95]. Indeed, a combination of these therapeutic approaches with existing anticancer therapies may increase therapeutic efficacy [6], as demonstrated by the successful combination of SST0001 with the monoclonal anti-VEGF antibody bevacizumab and the tyrosine kinase inhibitor sunitinib [96] or dexamethasone [97] in experimental anticancer therapy. Part of the success of these combinatorial approaches may be a result of the impact of currently used anticancer therapeutics on PG expression. For example, imatinib inhibits platelet-derived growth factor-mediated expression of HSPGs in breast cancer [98], whereas HS mediates the effect of trastuzumab in breast cancer cells [99]. Regarding the therapeutic utilization of miRNAs, these nucleic acids have been targeted using 2-Omethyl RNA oligonucleotides, locked nucleic acid-antimiRs and their cholesterol derivatives, or synthetic miRNA decoys, whereas their upregulation for therapeutic purposes can be achieved using lentiviral transfection techniques and, essentially, all of the related techniques previously utilized in gene therapy and small interfering RNA delivery [18,25,100]. To achieve a more efficient targeting of aberrantly expressed PGs and GAGs in malignant diseases, 5017

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miRNA-based approaches could be utilized. For example, radiation-induced angiogenesis in medulloblastoma involves both syndecan-1 and miR-494 [101], suggesting that a combinatorial approach may increase therapeutic benefit. A few studies in this direction have already shown promising results in preclinical models. For example, lentiviral miR-30-based RNA interference against heparanase was shown to abrogate melanoma metastasis with a favorable toxicity profile [102], whereas miR-155-based artificial miRNAs against heparanase inhibited melanoma cell adhesion, migration and invasiveness both in vitro and in vivo, associated with reduced chemokine expression and attenuated signaling via the p38-, Jnk- and Erk1/2-dependent pathways [103]. Finally, a new therapeutic perspective is indicated by the finding that cell surface HSPGs are important mediators of miRNA-containing exosome secretion [68] and internalization [54], suggesting that interference with cell surface HSPGs may have a profound impact not only on endogenous miRNA function in cancer, but also on therapeutic miRNA delivery [104,105].

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Acknowledgements Original research in the author’s laboratories on the topic of this review has been funded by the German Academic Exchange Service DAAD ‘Al Tawasul’, grants no. 57071624 and 56808461 (to SAI and MG), and by Science and Technology Development Funds (STDF), Egypt, grant no. 6309 (to SAI and HH). There are no conflicts of interest.

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SAI: performed literature searches and wrote the review; HH: performed literature searches, designed figures 1,2 & 4 and contributed to review writing; MG: performed literature searches, designed Figure 3, and wrote the review.

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FEBS Journal 281 (2014) 5009–5022 ª 2014 FEBS