Cancer Letters 347 (2014) 29–37

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

MiRNA in melanoma-derived exosomes Anna Gajos-Michniewicz a, Markus Duechler b, Malgorzata Czyz a,⇑ a b

Department of Molecular Biology of Cancer, Medical University of Lodz, Poland Department of Bioorganic Chemistry, Centre for Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland

a r t i c l e

i n f o

Article history: Received 17 December 2013 Received in revised form 21 January 2014 Accepted 3 February 2014

Keywords: Biomarker Exosome Melanoma MicroRNA MiRNA

a b s t r a c t Proteins, RNAs and viruses can be spread through exosomes, therefore transport utilizing these nanovesicles is of the great interest. MiRNAs are common exosomal constituents capable of influencing expression of a variety of target genes. MiRNA signatures of exosomes are unique in cancer patients and differ from those in normal controls. The knowledge about miRNA profiles of tumor-derived exosomes may contribute to better diagnosis, determination of tumor progression and response to treatment, as well as to the development of targeted therapies. We summarize the current knowledge with regard to miRNAs that are found in exosomes derived from tumors, particularly from melanoma. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Short characterization of biogenesis, structure and composition of exosomes Exosomes are small, intraluminal vesicles (30–150 nm in diameter), that were first described by Trams and coworkers [1]. Many cell types release exosomes, including reticulocytes, B and T cells, dendritic cells, mast cells and epithelial cells, as well as tumor cells [2–4] Exosomes have been detected in most body fluids [5,6]. They are composed of a lipid bilayer membrane containing ceramides, cholesterol, sphingolipids and phosphoglycerides [7–9]. Exosomes are enriched with a number of proteins, including members of the

Abbreviations: AGO-2, argonaute protein 2; BMDCs, bone marrow-derived cells; BMP4, bone morphogenetic protein 4; CCND1, cyclin D1; CDK4, cyclin-dependent kinase; DGCR8, DiGeorge critical region gene 8; EMT, epithelial to mesenchymal transition; ESCRT, endosomal sorting complexes required for transport; EZH2, histone-lysine N-methyltransferase; Hsp, heat shock protein; HMC-1, human mast cell line; JAK-STAT, Janus kinase, signal transducer and activator of transcription; KCNMA1, calcium ion-regulated potassium channel protein; MET, mesenchymal to epithelial transition; MITF, microphthalmia-associated transcription factor; MSCs, mesenchymal stem cells; MVBs, multivesicular bodies; MYLIP, myosin regulatory light chain-interacting protein; NHEMs, neonatal human epidermal melanocytes; NIK, NF-kappaB-inducing kinase; PTEN, phosphatase and tensin homolog; RBP1like, retinoblastoma binding protein 1 like; RISC, RNA-induced silencing complex; RUNX3, runt-related transcription factor 3; TD-exosomes, tumor derived exosomes; TLR, Toll-like receptor; Tregs, regulatory T cells. ⇑ Corresponding author. Address: Department of Molecular Biology of Cancer, Medical University of Lodz, 6/8 Mazowiecka Street, 92-215 Lodz, Poland. Tel./fax: +48 422725702. E-mail address: [email protected] (M. Czyz). http://dx.doi.org/10.1016/j.canlet.2014.02.004 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

tetraspanin family (CD9, CD81, CD82, CD63) and heat shock proteins (Hsp60, Hsp70, Hsp90), proteins participating in the biogenesis of the multivesicular bodies (annexins, Rab family GTPases, and ESCRT complex proteins) as well as interleukins and components of certain signaling pathways e.g. Wnt-b-catenin signaling proteins [10–13]. Several proteins present in exosomes are specific for the donor cells. Melanoma-derived exosomes contain an enhanced level of Melan A [14]. It is assumed that the lipoprotein content of exosomal membranes ensures the exosomal stability in the extracellular environment [15,16] and the ability to adhere to the target cells [17]. Exosomal biogenesis takes place in an endosomal compartment, called the multivesicular bodies (MVBs). Cytoplasmic RNA molecules and proteins are selectively packed into exosomes. Profiling of mRNA revealed that several hundred transcripts were enriched in exosomes in comparison to donor cancer cells [19]. Similarly, the miRNA profiles substantially differed between exosomes and cancer cells [18,20]. Recently, two short sequence motifs were identified which function as exosomal packaging signals for miRNAs, whereas three other motifs were identified which were not found in miRNAs from exosomes but in those retained inside the cell [21]. The exosomal packaging signals used to control miRNA sorting into exosomes were specifically recognized by sumoylated heterogeneous nuclear ribonucleoproteins, mainly hnRNPA2B1 and hnRNPA1. Exosomes are released into the extracellular space by exocytic fusion with the plasma membrane [22–26]. Their secretion is considered to be a highly organized and controlled process stimulated by various chemical, biological or mechanical factors. It has been

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demonstrated that heparanase, an enzyme up-regulated in many tumor cell lines is involved in the secretion of exosomes [27]. The exosomal secretion could be induced by c-irradiation followed by DNA damage [28], or by treatment with statins or calcium ionophores [8,29]. It has also been shown that mechanical detachment of breast cancer cells from various surfaces increases the exosomal secretion [30]. Moreover, low pH [31] or hypoxic conditions in cultures of melanoma or breast cancer cell lines, respectively, leads to a significant enhancement of exosomal release [32]. Secreted exosomes are taken up by the target cells through three possible mechanisms. The first one comprises simple fusion with the cellular membrane, directly releasing the content of the vesicles into the cytoplasm [17,33]. The second one assumes exosomal uptake by endocytosis [5,34]. In the third one, the uptake is a non-random process, but it is dependent on the presence of distinct receptor proteins that enable the binding of exosomes to the target cells [35]. A general scheme of exosomal biogenesis, the release from the cell of origin as well mechanisms of exosomal uptake by the target cell are presented in Fig. 1 and more information can be found in several recently published review articles [35–39]. Exosomes were originally thought to play a main role in cellular debris disposal, but nowadays their role in long distance cell–cell communication is increasingly acknowledged as they function as important transporters of miRNAs, piRNAs, lncRNAs, rRNAs, snRNA, snoRNAs, tRNAs, mRNAs, DNA fragments, and proteins [40,41]. According to the Exocarta database (http://www.exocarta.org) which gathers information about the contents of exosomes, 4563 proteins, 1639 mRNAs, 764 miRNAs and 194 lipids have already been identified in exosomes from different cell types of multiple organisms.

2. Exosomes as miRNA carrying vesicles Exosomal transfer of mRNAs and miRNAs has been recognized as an important cellular communication system for the exchange of genetic and epigenetic information between cells [5,40,42]. MiRNAs (microRNAs) are small (19–25 nt), non-coding regulatory RNAs. The miRBase database which contains information for

miRNAs (release 20, http://www.mirbase.org) lists 24521 entries representing hairpin precursor miRNAs, and 30424 mature miRNA products from 206 species. Among them are 1872 miRNAs from Homo sapiens. MiRNA biogenesis (Fig. 2) begins with their transcription by RNA polymerase II generating primary miRNAs (pri-miRNA) comprising a hairpin stem, a terminal loop and two single stranded regions. Pri-miRNAs are processed in the nucleus by DROSHA and DGCR8 (DiGeorge Critical Region gene 8) that is responsible for the precise pri-miRNA cleavage. This process generates short hairpin structures, the pre-miRNAs, which are exported from the nucleus by exportin-5. The maturation of pre-miRNAs occurs in the cytoplasm through cleavage by DICER. The mature miRNA is then incorporated into a protein complex called RISC (RNA induced silencing complex), where the miRNA meets its complementary target mRNAs. It is assumed that nucleotides 2–8 of the miRNA, called the seed region, must bind contiguously to a complementary sequence on the target mRNA lying in the 3’-UTR region. Whether miRNA promotes mRNA degradation or translation depends on the degree of complementarity. Perfect or near-perfect complementarity beyond the seed region sequence induces mRNA degradation, whereas imperfect binding results in translational attenuation [43–47]. The multistage maturation process of miRNAs is strictly regulated by many factors, and its deregulation leads to impairment of miRNA expression, giving rise to progression of various diseases [48]. Epigenetic mechanisms are crucial for controlling miRNA expression [49,50]. Several intronic miRNAs are regulated along with their host protein-encoding genes. For example, miR211 that functions as a melanoma tumor suppressor is an intronic miRNA coexpressed with its host gene, melastatin [51]. Intergenic miRNA expression can also be regulated like protein-encoding genes by DNA methylation or histone modifications [52,53]. Moreover, regulatory circuits may arise through miRNAs that repress the translation of enzymes that are crucial for epigenetic remodeling processes, and this may contribute to the epigenetic control of miRNA synthesis [54]. Some evidence suggests that miRNAs may also exert direct epigenetic functions at the promoter regions through modulating transcription. Mediated by AGO proteins, miRNAs target gene promoters containing complementary

Fig. 1. Exosomal biogenesis and mechanisms of exosomal uptake by target cell. Exosomes, small vesicles formed by inward budding, contain miRNAs, mRNAs and proteins. They are released from the multivesicular body (MVB) when MVB fuses with the plasma membrane. Alternatively MVB can be degraded in lysosomes. Released exosomes can be directly fused to the target cell, releasing its content into the cytoplasm, or they can be endocytized. The third mechanism is the juxtacrine signaling that involves specific receptor proteins that enable the binding of exosomes to the target cells.

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Fig. 2. MiRNA biogenesis. MiRNA is transcribed in the nucleus by polymerase II, generating pri-miRNA, that undergoes DROSHA processing, generating harpin loops of about 70 nucleotides (pre-miRNA). The pre-miRNA is then exported into the cytoplasm by EXPORTIN-5 and processed by DICER into the miRNA duplex. One of the strands is released and degraded by AGO protein, whereas the second is incorporated into a multiple-protein nuclease complex, the RNA-induced silencing complex (RISC), which regulates protein expression. The mode of regulation, translation attenuation or mRNA degradation, depends on the degree of complementarity beyond the seed region (SR).

sequences to recruit chromatin remodeling complexes [55,56] or RNA polymerase 2 [57]. MiRNAs are able to influence the expression of multiple genes within the cell of origin or can affect the gene expression in neighboring cells or even distant cells due to their transport through the circulation [10,58,59]. The extracellular transfer of miRNA can be mediated by protein transporters [60,61] or by exosomes [62]. It is worthy to point out that encapsulation of miRNAs within the exosomal structure ensures their stability and provides protection against RNase digestion and other environmental damage [5,10]. Taylor and Gercel-Taylor [63] examined storage stability of exosomal miRNAs derived from serum of women suffering from ovarian cancer. Comparison of miRNA intensities on microarrays in samples stored at 4 °C for up to 96 h and at 70 °C for up to 28 days indicated that exosomal miRNAs were stable and did not significantly change during storage in both conditions [63]. The first study demonstrating the exchange of nucleic acids between cells via exosomes was done by Valadi et al. [64]. The exosomal transfer between mouse (MC/9), and human mast cell lines (HMC-1), resulted in the expression of murine proteins in human cells. The association of miRNAs with exosomes has been demonstrated by inhibition of vesicle release. As exosomes are secreted through a ceramide-dependent mechanism [26], treatment with an inhibitor of a sphingomyelinase 2 prevented the cleavage of sphingomyelin into ceramide and resulted in a dose-dependent reduction of extracellular miRNAs, while their cellular expression stayed unchanged. Moreover, decreasing the protein level of sphingomyelinase 2 by siRNA transfection resulted in the attenuation of extracellular miRNA levels, whereas overexpression of sphingomyelinase 2 induced the secretion of miRNAs from HEK293 (Human Embryonic Kidney 293) cells [26].

3. MiRNAs and exosomes in tumor development Aberrant miRNA expression is observed in many cancers when compared to their normal tissue counterparts [47,65]. A possible role of miRNAs in cancer development was postulated when the influence of miRNAs on proliferation and apoptosis was observed

in Caenorhabditis elegans and Drosophila [66,67]. The expression of miRNAs in tumors is frequently dysregulated and miRNAs can function as oncogenes or tumor-suppressor genes [68]. Among the mechanisms leading to aberrant miRNA expression, genetic abnormalities, epigenetic alterations, as well as aberrant miRNA biogenesis can be distinguished. Genetic alterations of miRNAs comprise mostly structural rearrangements, loss and polymorphisms [69,70]. For example, deletion of miR-17-92 cluster was frequent in melanomas, ovarian, and breast cancers [69]. Epigenetic alterations are associated mainly with hypermethylation or hypomethylation in miRNA promoter regions [48,71]. For example, it was demonstrated that expression of miR-18b was substantially reduced in melanoma specimens and cell lines by hypermethylation of its promoter, which was accompanied by overexpression of the target transcript, MDM2 [72]. In contrast to melanocytes and keratinocytes, melanoma cells exhibit hypermethylated miR375 promoter [73], and overexpression of miR-375 altered the cell morphology and attenuated proliferation and invasion of melanoma cells. Demethylation or methylation of 50 -CpG islands in the promoter region of miR-200 were associated with the epigenetic control linked to epithelial to mesenchymal transition (EMT) or mesenchymal to epithelial transition (MET) phenotypes. According to Davalos et al. [74], 50 -CpG islands in the promoter region of miR-200 were unmethylated in human cancer lines with epithelial features, whereas CpG island hypermethylation and associated silencing was observed in transformed cells with mesenchymal characteristics. Moreover, the plasticity of the process contributes to the evolving and adapting phenotypes of cancer cells [74]. Already earlier the miR-200-ZEB1-E-cadherin axis was identified as crucial for EMT and mutual repression between ZEB1 and the miR-200 family promote EMT in cancer cells [75,76]. Several other diversely regulated miRNAs involved in the regulation of EMT during cancer progression were identified. Most recently, a double-negative feedback loop involving AP4 and p53-induced miR-15a/16-1 to regulate EMT and metastasis has been identified in colorectal cancer [77]. In addition to genetic and epigenetic alterations, there are various dysregulations of miRNA processing associated with mutations of co-factors or overexpression of proteins involved in miRNA biogenesis including DICER, DROSHA and

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Fig. 3. The role of miRNA in the intercellular transfer of information. The first level of regulation of the information transfer is the selection of miRNAs to be expressed in the cell (1) and different miRNAs are expressed in normal and cancer cells. The second level is the selective packaging of exosome content (2) by mechanisms that just started to be elucidated [21]. The miRNA profiles substantially differ between exosomes and cancer cells [18,20]. Microenvironmental stimuli such as hypoxia or low pH influence the exosome secretion (3). MiRNAs from tumor-derived exosomes are mediators of intercellular communication. They influence proliferation rate and chemoresistance of other cancer cells (4), modify normal cells in the local microenvironment (5) and distant organs (6). For example, melanoma-derived exosomes can promote migration of endothelial cells and affect angiogenesis by transferring miR-9 to endothelial cells [79]. Exosomes released from melanoma cells in hypoxic conditions promote metastatic niche formation by ‘educating’ bone marrow-derived cells towards a pro-metastatic and pro-vasculogenic phenotype [82]. Tumor-derived exosomes can prepare the premetastatic niche by targeting non-transformed cells of premetastatic organs with miRNA [83]. Melanoma exosomes home to sentinel lymph nodes where they recruit melanoma cells [84]. The profiling of exosomal miRNAs in the body fluids might be used as a substitute for tumor tissue miRNA analysis. DC, dendritic cells; EC, endothelial cells; T, lymphocyte T; BMDCs, bone marrow-derived cells.

AGO-2. These disturbances result in differential miRNA processing as well as improper cellular localization. Another mechanism of miRNA regulation promoting cancer is a direct interaction between two different miRNAs through sequence match. For example, it was demonstrated that formation of miR-107: let-7 duplex reduced the tumor suppressive effects of let-7 [78]. The dysregulation of miRNAs in cancer tissue directly or indirectly influences the intercellular exchange of various proteins and genetic material via exosomes [4]. A positive correlation between the abundance of secreted exosomes, and cancer stage and progression rate was observed [15]. Although Taylor and GercelTaylor [63] found no significant differences in exosomal miRNA expression profiles between patients with early versus late stage of ovarian cancer, these miRNAs levels were significantly elevated comparing to miRNA expression in exosomes derived from benign disease [63]. The transfer of miRNAs via exosomes may contribute to tumor progression through the transfer of genetic information not only between closely localized cells but also to distant tissues. Possible modifications of tumor cells, cells in the tumor microenvironment and distal organs by miRNA from melanoma-derived exosomes are shown in Fig. 3. In the direct tumor microenvironment, hypoxia is one of the factors contributing to the increased release of exosomes, and hypoxic tumors are generally characterized by a more aggressive phenotype and rather poor prognosis [32]. Exosomes released under hypoxic conditions contribute to the stimulation of angiogenesis [15]. Melanoma cell-derived exosomes can promote migration of endothelial cells and affect angiogenesis by transferring miR-9 to endothelial cells, which activates the JAKSTAT pathway [79]. Tumor derived exosomes (TD-exosomes) may also support the tumor dissemination by exerting immunosuppressive function. For example, miRNAs from lung cancer-derived exosomes were able to bind to the Toll-like receptors (TLRs) in immune cells and induced a prometastatic inflammatory

response [80]. Tumor cells by employing exosomes can also educate distant tissues for tumor cell hosting [81,82]. The preparation of the premetastatic niche by TD-exosomes is thought to occur through targeting non-transformed cells of premetastatic organs with miRNA [83]. For example, TD-exosomes released in hypoxic conditions ‘educated’ bone marrow-derived cells (BMDCs) towards a pro-metastatic and pro-vasculogenic phenotype via increasing the level of the MET oncoprotein [82]. In other experiments it has been shown that melanoma exosomes first home to sentinel lymph nodes where they recruit melanoma cells [84]. Although TD-exosomes are considered to be pro-tumorigenic, it has also been found that some exosomes possess anti-tumorigenic abilities. Exosomes isolated from pancreatic cancer cells were shown to induce apoptosis of cancer cells [5]. Exosomes may also present tumor antigens to dendritic cells stimulating in this way an antitumor response [85]. It was demonstrated in a murine melanoma model that TD-exosomes could activate NK cells, which was associated with reduced primary tumor size and metastases [86]. 4. Exosomal miRNA in melanoma Melanoma develops by the malignant transformation of melanocytes and is considered as the most aggressive form of skin cancer characterized by a high mortality rate. For the improvement of therapies it is necessary to determine the complex molecular mechanisms leading to melanoma. There is no doubt that miRNAs, as crucial post-transcriptional regulators of gene expression, are important components in melanoma biology [87,88]. MiRNAs deregulate relevant transcription factors to control the development and metastasis of malignant melanoma [71,89,90]. It was reported that cancer growth is facilitated by the loss of certain miRNAs, whereas cancer progression is promoted by overexpression of other miRNAs [91]. Xiao and coworkers investigated the

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miRNA expression profiles of melanoma cell lines (A375) and their exosomes in comparison to those of normal melanocytes (HEMaLP) [92]. Exosomal yield was higher in the melanoma cell line than in normal melanocytes and substantial differences in exosomal miRNAs expression profiles between A375 and HEMa-LP exosomes were observed [92]. Xiao and coworkers identified 130 miRNAs up-regulated and 98 miRNAs down-regulated in melanoma cellderived exosomes versus melanocyte-derived exosomes, and 70 miRNAs were associated with cancer. Several of them were recognized before as crucial contributors to melanoma genesis and progression (Supplementary Table S1). Members of miRNA let-7 family were the first miRNAs shown to be involved in the processes of carcinogenesis. Their importance for melanoma development was confirmed especially for let-7a and let-7b [93]. Transcripts of integrin b-3 and cyclin D1 are among targets of let-7a and let-7b, respectively. Let-7a regulates the expression of integrin b-3 by direct interaction with the 3’UTR of its mRNA. Integrin b-3 promotes the progression of melanoma, enhancing its migratory and invasive potential. It was reported that the loss of let-7a expression resulted in higher integrin b-3 levels in melanoma cells. Let-7a regulates the expression of RAS; thereby its loss enhances cellular proliferation [93]. MiRNA let-7b is assumed to be a direct or indirect cell cycle regulator in melanoma. Schultz et al. [94] demonstrated that let-7b directly interacts with the cyclin D1 3’UTR, whereas the effects on other cyclins (A, D3) and CDK4 are considered to be indirect. Experiments employing let-7b overexpression in melanoma cells resulted in the significant decrease of cyclins D1, D3, and Cdk4 protein expression. Thus, let-7b is considered to be a negative regulator of melanoma growth and proliferation [94]. Microphthalmia-associated transcription factor (MITF) is an important regulator of melanocyte differentiation and transformation [95]. This transcription factor plays a crucial role in the development of melanocytes as well as in progression and plasticity of melanoma. In melanocytes, MITF is considered to be involved in important cellular programs such as differentiation, cell survival, cell cycle, cellular motility and miRNA biogenesis [71]. As MITF is of central importance in the biology of melanocytes, its regulation, mediated by miRNAs was investigated. miR-101 was identified as capable of directly targeting the MITF transcript [96]. Segura et al. [97] determined miR-182 as a negative regulator of MITF expression and showed that miR-182 promotes migration and survival of melanoma cells by down-regulating MITF expression. Moreover, they demonstrated that overexpression of miR-182 stimulated oncogenic properties of melanoma cells, mainly anchorage independent growth and movement through the extracellular matrix [97]. MiR-221 and miR-222 were also shown to be dysregulated in melanoma. It has been demonstrated that they participate in the regulation of tumor proliferation, migration, invasion and apoptosis [98]. Felicetti et al. [99] showed that upregulation of miR-221/222 expression during melanoma progression resulted in diminished expression of c-Kit, a transmembrane receptor with tyrosine kinase activity important for melanogenesis [99]. Igoucheva and Alexeev [100] provided evidence that c-Kit activity is regulated mainly by miRNA-dependent mechanisms. Furthermore, it was reported that overexpression of miR-221 and miR-222 in melanoma cells characterized by low endogenous levels of both miRNAs, resulted in increased cellular proliferation, invasiveness as well as chemotactic capabilities. On the other hand, treatment of melanoma cells harboring high levels of miR-221 and miR-222 with antagomiRs against both miRNAs led to a reduction of the proliferation rate as well as a decrease of invasiveness and migration [99]. According to the current literature, miR-31 acts either as an oncomiR or as a tumor-suppressive miR in various tumors [101]. Asangani et al. [101] demonstrated that melanocytes were

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enriched with miR-31 and the progression towards melanoma caused miR-31 abrogation. They proved that miR-31 is a tumor suppressor targeting multiple oncogenes in melanoma including: the non-receptor protein tyrosine kinase SRC, the proto-oncogene MET, NF-kappaB-inducing kinase (NIK) and EZH2 (histone-lysine N-methyltransferase). The loss of miR-31 expression was associated with genomic deletions on chromosome 9p21 encoding miR-31. Deletions in this chromosome are characteristic for melanoma. Alternatively, the loss of miR-31 expression in melanoma could also be attributed to epigenetic silencing by DNA methylation and EZH2-mediated histone methylation. MiR-31 exerts inhibitory effects on cell motility and invasion. Moreover, miR-31 creates a mutually antagonistic loop with EZH2 as the loss of miR-31 expression is associated with EZH2 overexpression leading to melanoma progression and EZH2-mediated further down-regulation of miR-31 through chromosome silencing [101]. Another target gene for miR-31 is the melanoma specific oncogene RAB27a that is involved in the regulation of melanosome trafficking and exosome formation. Deregulated miRNA expression in melanoma is also found in the miR-106-363 group. MiR-106-363 is considered as an oncogenic miRNA gene cluster, whose members regulate genes involved in cellular attachment, motility, cell contact inhibition and proliferation. Mueller et al. [102] identified miRNAs belonging to the miR-106-363 cluster, whose up-regulation leads to metastatic melanoma progression. Among the target genes are myosin regulatory light chain-interacting protein (MYLIP) and retinoblastoma binding protein 1 like (RBP1-like). Comparison of the expression profiles of miRNAs in tumor cell lines, versus normal melanocytes, revealed a strong up-regulation of miR-106a and miR-92 and a rather slight up-regulation of miR-19b [102]. Another miRNA important for melanoma biology, whose overexpression is also reported in other cancer types, is miR-21 [103]. MiR-21 is up-regulated in melanoma cell lines, where it acts as oncogene, compared to benign melanocytic cells. One of the most prominent target genes of miR-21 is PTEN (phosphatase and tensin homolog). PTEN antagonizes the phosphatidylinositide 3-kinase (PI3K) pathway and down-regulation of this phosphatase increases signaling through PI3-K and Akt. PI3-K/Akt signaling promotes proliferation and survival of melanoma cells and activating mutations of this pathway has been shown to confer resistance to BRAF inhibitorbased chemotherapy [104]. Down-regulation of miR-21 in melanoma cell lines, harboring high endogenous miR-21 expression, induced apoptosis. In melanoma patients, enhanced levels of miR-15b were associated with poor prognosis. Ectopic downregulation of miR-15b resulted in reduced proliferation and enhanced apoptosis of melanoma cells and melanoma cell lines originally presenting high miR-15b expression [105]. Bar-Eli [106] and Penna et al. [107] reported up-regulation of miR-214 in melanoma cell lines and tissue samples where it targeted the transcripts of integrin alpha-3 and transcription factor AP-2 gamma. It was reported that its overexpression stimulated motility and promoted survival of melanoma cells as well as invasion and formation of metastases [106,107]. MiR-30b and miR-30d tended to be overexpressed in melanoma and this correlated with malignancy and poor survival [108]. Overexpression of miR-30b/30d promoted invasiveness of melanoma cells in vitro and increased their metastatic potential in vivo, by suppressing expression of GalNAc transferase 7 (GALNT7). Down-regulation of GALNT7 expression in melanoma cells increased the production of IL-10, a highly immunosuppressive cytokine. Furthermore, miR-30d suppressed T-cell activation and increased the recruitment of Tregs into metastatic tissues thus contributing to the inhibition of immune surveillance [108]. Melanoma progression is also regulated by miR-532-5p. Kitago et al. [109] observed up-regulation of cellular expression of miR-532-5p when they examined metastatic melanoma cell lines compared with normal melanocytes, in which it

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targets Runt-related transcription factor 3 (RUNX3). Down-regulation of the tumor suppressor gene RUNX3 contributes to tumor progression. The inhibition of miR-532-5p resulted in up-regulation of RUNX3 mRNA and protein in melanoma cell lines [109]. All miRNAs described so far as upregulated in melanoma cells were also tested in the exosome miRNA profiling study of Xiao et al. [92]. For most of them (let-7a, miR-182, miR-221, miR-222, miR-31, miR-19b-2, miR-20b and miR-92a-2, miR-21, miR-15b, miR-210, miR-30b, miR-30d, and miR-532-5p) enhanced levels were found in exosomes released from A375 melanoma cells in comparison to their levels in exosomes derived from normal melanocytes. However, the levels of let-7b and miR-214 were lower in A375 exosomes than in exosomes from normal melanocytes suggesting that besides a set of conserved changes also individual differences in the exosomal miRNA profile exist between particular cell lines and probably tumors. Several miRNAs are down-regulated in different types of cancers including melanoma. MiR-125b is considered to be one of the representatives of this group. According to Glud et al. [110], down-regulation of miR-125b expression was associated with a decrease in spontaneous apoptosis, whereas its ectopic expression induced senescence. Kappelmann et al. [90] demonstrated that cJun expression, a main regulator of melanoma progression, was regulated post-transcriptionally by miR-125b. Their experiments showed a reduced miR-125b expression in malignant melanoma cell lines and tissue samples compared with melanocytes. Furthermore, the treatment of melanoma cells with pre-miR-125b, resulted in strong suppression of cellular migration and proliferation [90]. Another study, comparing the miRNA expression in melanoma cell lines to normal melanocytes (NHEMs), observed down-regulated expression of miR-148a in primary tumor cell lines [80]. Experiments of Haflidadóttir et al. [111] showed that miR-148 negatively affected MITF mRNA levels in melanoma cells through conserved binding sites in the 3’UTR sequence. They demonstrated that the transfection with anti-miR-148 blocked its effect on endogenous MITF mRNA. Mazar et al. [112] pointed out that miR-211 directly targeted the transcript of KCNMA1, a calcium ion-regulated potassium channel protein. Reduced expression of miR-211, in comparison to melanocytes, resulted in increased expression of the target transcript of KCNMA1. MiR211 is encoded in an intron of the KCNMA1 and thus co-regulated by MITF whose activity is required for high KCNMA1 expression [71,112]. The expression of KCNMA1, contributes to high cellular proliferation and increased invasiveness of melanoma cell lines [112]. Another miRNA that tends to be down-regulated in melanoma is miR-193b that was recognized as an inhibitor of melanoma cell proliferation directly targeting the transcript of cyclin D1 [113]. MiR-196a comprising miR-196-a-1 and miR-196-a-2 was shown to be down-regulated in melanoma cell lines [114]. This was accompanied by the enhanced expression of HOX-C8 and HOX-B7. The induction of basic fibroblast growth factor by HOX-B7 raises Ets-1 activity which leads to the induction of bone morphogenetic protein 4 (BMP4) expression. As BMP4 is considered to be a modulator of migration in melanoma, its overexpression contributes to disease progression [114]. Members of the miR-200 cluster are also considered to be important in melanoma development. According to Adam et al. [115] and ElsonSchwab et al. [116], all members of the miR-200 family have been implicated as crucial modulators of EMT that plays an important role in tumor migration, invasion and metastasis. Deregulation of miR-200 expression has been observed in various cancer types [116]. In melanoma, expression of several members of the miR200 group comprising miR-200a, miR-200b, miR-200c, miR-429 and miR-141, was shown to be down-regulated [113,117] or upregulated [116]. Another miRNA whose expression was reduced in melanoma cell lines is miR-205 which targets E2F1 and E2F5.

Dar et al. [118] showed that ectopic overexpression of miR-205 caused the inhibition of melanoma proliferation and induced senescence [118]. Among the miRNAs down-regulated in melanoma cells, miR-148a, miR-211, miR-200b, miR-429 and miR-205 showed the same trend in the exosomal profiling study [92]. Only the levels of miR-125b, miR-193b and miR-196a in A375 exosomes were higher compared to their level in exosomes of normal melanocytes.

5. Exosomes as delivery system There is great interest in exosome mediated transport, focusing on their role in delivery of their content across biological membranes [119–124]. As exosomes are autologously generated within the host, they can become the non-immunogenic carriers of drugs, RNA or target proteins. Therefore, exosome-based therapies may become an attractive strategy against cancers and other diseases [10]. A study by Alvarez-Erviti et al. showed that systemically applied exosomes that are capable of penetrating the blood–brain barrier, could be used for the targeted delivery of siRNA into the brain in vivo [125]. Direct transport of membrane proteins by exosomes has been reported by Ruiss et al. [126]. Exosomes were used for the transfer of the CD154 protein to T cells to improve the interaction of helper T cells with B-CLL (B cell chronic lymphocytic leukemia) cells through increased CD154 binding to CD40. Targeting B cells was achieved by incorporation of gp350, the major envelope protein of Epstein–Barr-Virus, into the exosome membrane. In a murine xenograft breast cancer model, let-7a miRNA was delivered to the tumor cells by intravenously injected exosomes [127]. The miRNA was introduced into exosomes by lipofection and the targeting of the tumor cells was achieved by expression of a EGFRbinding peptide in the exosome membrane. Most recently, it has been shown that intra-tumor injection of exosomes derived from miR-146-transfected marrow stromal cells significantly reduced glioma xenograft growth in a rat model [128], among the miR-146b-down-regulated transcripts were those for EGFR and NF-jB. Escudier et al. [129] reported the usage of autologous exosomes obtained from monocyte-derived dendritic cell cultures, that were pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients in a phase I clinical trial. Their results emphasized the feasibility of large scale exosome production and the safety of exosome administration, as there was no grade II toxicity detected [129]. At present, there are several exosomebased clinical trials in different kinds of cancers. miRNA-based trails involving exosomes have not been initiated yet [10]. Natural exosomes, which deliver their cargo to the cytosol of recipient cells, demonstrate several advantages over synthetic carriers. However, some technical problems concering production, purification and loading with siRNA still remain [130]. Many cell types can secrete exosomes, however, the only cell type known to have scalable capacity to produce exosomes is the mesenchymal stem cell (MSC) [131]. MSCs are distributed among perivascular niches of various tissues and represent a tissue-specific functional biodiversity [132]. This ethically noncontroversial source of exosomes can be easily isolated and expand ex vivo, however, this expansion is not infinite without genetic manipulation. Nevertheless, MSCs are considered as an efficient mass producer of exosomes [131]. It should be noted, however, that natural bone marrow MSCexosomes was found to favor tumor growth and angiogenesis, and this role was partially related to the increased expression of VEGF in tumor cells [133]. Delivery vehicles have to fulfill two basic requirements: they have to be loaded efficiently with the molecules to be transferred and they should contain a targeting structure on their surface for specific binding to the target cells. Exosomes can be loaded and are amenable to membrane modification that can

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enhance cell type specific targeting. Moreover, by assembly of liposomes harboring crucial components of natural exosomes, functional exosome mimetics may be created as a novel class of drug delivery systems [123]. Thus, exosomes can be manipulated in several ways to satisfy both requirements, loading and targeting. 6. Future directions MiRNAs found in exosomes are characteristic for the cell from which these nanovesicles are released [5]. MiRNA signatures of circulating exosomes in cancer patients differ from those in normal controls [134]. Therefore, exosomal miRNAs might serve as diagnostic markers of cancer [5,46,63,135], including melanoma [82]. Furthermore, due to the simplicity of isolation techniques of circulating exosomes, exosomal miRNA profile analysis might become a non-invasive tool for the detection of cancer [63,135] even in early stages of the disease. Further studies are needed to confirm and extend the prognostic and diagnostic potential of exosomal miRNA profiles. The accumulating knowledge about exosomes may have potential application in monitoring patient response to anticancer treatment. Exosomes may be also exploited as therapeutic gene or drug delivery systems. The potential of exosomes as therapeutic delivery vehicles has been already shown and it can be further expanded by utilizing their ability to penetrate the blood–brain-barrier and their intrinsic homing capability. Thus, the knowledge about miRNA, mRNA and protein profiles in exosomes released from melanoma and other tumors may contribute to better diagnosis, determination of tumor progression and response to treatment, as well as to further development of targeted therapies. Conflict of Interest

[8]

[9]

[10]

[11] [12] [13]

[14]

[15]

[16] [17]

[18]

[19]

[20]

None declared. [21]

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