Table of Contents

    Topic review

    Non-Coding RNA in Melanoma

    Subjects: Oncology
    View times: 57

    Definition

    Considered to be highly lethal if not diagnosed in early stages, cutaneous malignant melanoma is among the most aggressive and treatment-resistant human cancers, and its incidence continues to rise, largely due to ultraviolet radiation exposure, which is the main carcinogenic factor. Over the years, researchers have started to unveil the molecular mechanisms by which malignant melanoma can be triggered and sustained, in order to establish specific, reliable biomarkers that could aid the prognosis and diagnosis of this fatal disease, and serve as targets for development of novel efficient therapies. The high mutational burden and heterogeneous nature of melanoma shifted the main focus from the genetic landscape to epigenetic and epitranscriptomic modifications, aiming at elucidating the role of non-coding RNA molecules (ncRNAs) in the fine tuning of melanoma progression. Studies have shown that ncRNAs, among them microRNAs and lncRNAs, play a role in melanoma invasion, metastasis and acquired resistance to treatment. In addition, they could serve as prognostic/diagnostic biomarkers and potential targets for promising therapeutic strategies. 

    1. Introduction

    Non-coding RNAs (ncRNAs) have been observed to tamper with the efficiency of molecular therapies, in addition to facilitating melanoma invasion and metastasis [1][2]. They represent important epigenetic and epitranscriptomic regulators, classified and differentiated into long (lncRNAs) and small (sncRNAs) molecules, according to their length which can be longer or smaller than 200 bps. While multiple types of RNAs can be found in the small category, particular interest has been devoted to studying the deregulation of microRNAs (miRNAs/miR) in melanoma [3]. miRNAs can modulate the gene expression at post-transcriptional level mostly by interfering with the 3′UTR region of mRNAs, and substantial evidence points to their involvement in each stage of melanoma progression [1][4]. In contrast, the epigenetic control exerted by lncRNAs depends on the recruitment of regulatory proteins at specific DNA target regions, to silence or activate gene promoters, and their role in malignant melanoma is more elusive [5][6]. Some lncRNAs have been described to stand as decoys or sequesters of miRNAs, while others play a role in stabilizing the translational ribosomal machinery [7].

    2. MicroRNAs Modulate Melanoma Invasion and Metastasis

    The role of miRNAs in melanoma progression is very tangled, therefore excellent reviews can be consulted here: [1][8][9][10]. In principle, miRNAs influence the evolution of melanoma to secondary sites mainly by: (A) regulating the expression of MITF-M, an essential melanocyte-and melanoma-specific transcription factor that operates as a “switch” in the establishment of an invasive or proliferative phenotype; (B) remodelling the extracellular matrix (ECM); (C) promoting epithelial-to-mesenchymal transition (EMT) and its reverse, mesenchymal-to-epithelial transition (MET); and (D) preparing the formation of the pre-metastatic niche [1][9][11][12][13] (Figure 1).

    Figure 1. The role of non-coding RNAs in melanoma invasion and metastasis: (A) Both miRNAs and lncRNAs modulate the expression of MITF-M, directly or indirectly, therefore influencing the switch towards an invasive or proliferative phenotype; (B) The deregulated expression of ncRNAs facilitates ECM remodelling, by targeting integrins, increasing matrix metalloproteinases (MMPs) and diminishing the metallopeptidase inhibitors (TIMPs), therefore promoting ECM degradation and decreasing tumour cell-matrix adhesion; (C) Multiple miRNAs and lncRNAs favour the EMT-like process of melanoma cells, which enhances their migratory capacity, mainly by affecting the expression of specific adhesion molecules. Some ncRNAs also promote the MET-like process of malignant melanocytes, which leads to enhanced proliferation and colony formation, thus favouring metastasis; (D) The formation of a pre-metastatic niche at distant sites is mainly influenced by melanoma-derived exosomes, enriched in miRNA cargo (e.g., miR-155, miR-210, miR-214). In sentinel lymph nodes, they instigate microenvironmental changes that facilitate the recruitment, trapping and growth of circulating tumour cells. They also prime bone-marrow-derived cells (BMDCs), which achieve a vasculogenic, metastatic phenotype, and facilitate their recruitment to distant sites, where BMDCs will contribute to the formation of a pre-metastatic niche. LncRNA HOTAIR is enriched in lymph node metastases compared to primary tumours, which may suggest a role in the pre-metastatic niche formation. POU3F2: POU-domain class 3 transcription factor 2; NEDD9: Neural precursor cell expressed developmentally down-regulated protein 9; ZEB1: zinc finger E-box-binding homeobox 1. Colour code: red for upregulation and green for downregulation. Figure created with BioRender.com.

    Reduced expression of MITF-M in melanoma cells determines the acquisition of an invasive phenotype [12]. Several miRNAs have been found to regulate the activity of this lineage-restricted gene at a post-transcriptional level, among them miR-182, miR-137, miR-211 and miR-107 [13][14][15][16][17]. Overexpression of miR-182 stimulates melanoma cell migration and invasion through the direct downregulation of MITF and FOXO3 expression; studies on melanoma cell lines and tissue samples have found that its expression increases gradually from primary to metastatic stage [15]. Similarly, high levels of miR-137 expression have been correlated with poor survival in advanced melanoma patients [18]. In contrast, decreased expression of miR-211 can be observed in highly invasive melanoma cell lines [19]. Normally, miR-211 prevents the loss of cell adhesion through the negative modulation of NUAK1 [20], thereby inhibiting the migratory and invasive capacity of melanoma cells [21]. Additionally, it has been proposed that miR-211 can transcriptionally repress POU3F2 (POU-domain class 3 transcription factor 2), also known as BRN2 (brain-specific homeobox 2), which is a known suppressor of MITF. As such, loss of miR-211 can increase the expression of BRN2, and therefore inhibit MITF, ensuring that malignant melanocytes are kept in a pro-invasive, dedifferentiated state [16]. Recently, Zhao et al. reported the downregulation of miR-107, another tumour suppressor that inhibits melanoma cell invasion by targeting POU3F2 [17]. In BRAF-mutant cells, overexpression of BRN2 contributes to cytoskeletal rearrangement and increased cell invasion [22].

    Several miRNAs play a role in ECM remodelling [23]. In melanoma, loss of let-7a, which is a negative regulator of ITGB3, leads to elevated levels of integrin α3 [24], while suppression of let-7b indirectly enhances the production of matrix metalloproteinase (MMP)-9 [25], facilitating collagen degradation and cell invasion. Meanwhile, miR-21 is thought to induce an invasive phenotype in melanoma cells by targeting the mRNA of tissue inhibitor of metalloproteinases (TIMP)-3 [26]. Lastly, the increased expression of miR-30d/miR-30b cluster stimulates the invasive and metastatic potential of melanoma cells, both in vitro and in vivo, potentially by silencing GALNT7 (polypeptide N-acetylgalactosaminyltransferase 7), which strongly affects the O-glycosylation of membrane proteins, and subsequent interaction with the ECM [27].

    True EMT in melanoma is not possible as melanocytes are not epithelial cells, however some miRNAs are implicated in an EMT-like process that promotes the invasion and metastasis of tumour cells. One of them is the cluster miR-224/miR-452, which silences a metastatic suppressor that normally inhibits E2F1, and which has been shown to facilitate the cytoskeletal rearrangement of less aggressive cells, resulting in increased migratory and invasive propensity [28]. Also considered of particular relevance, the miR-200 family (miR-200a, miR-200b, miR-200c, and miR-141) drives the EMT-like process by upregulating the expression of Bmi-1 oncogene, which in turn promotes the activation of PI3K/AKT and MAPK cascades. This negatively influences the expression of ZEB1 (zinc finger E-box-binding homeobox 1) and E-Cadherin, at the same time stimulating N-Cadherin and vimentin expression [29]. High levels of miR-214 have also been associated with enhanced cell motility and metastatic potential, as this specific miRNA indirectly downregulates the expression of some adhesion molecules (MCAM, E-cadherin) and metallopeptidase inhibitors (TIMP1, TIMP2) [30].

    The MET-like process of malignant melanocytes is favoured by miR-125b overexpression, who directly targets a transcript of NEDD9 (neural precursor cell expressed developmentally down-regulated protein 9) [31][32], and whose in vitro inhibition was found to decrease the invasive potential of aggressive melanoma cells [31]. Other miRNAs (miR-34b, miR-34c, and miR-199a-3p) also contribute to this mesenchymal movement by targeting the mRNA of tyrosine-protein kinase Met (c-MET), whose increased expression facilitates melanoma cell migration and metastasis [33][34].

    miRNAs also participate in the formation of a pre-metastatic niche at distant organs that will enable the implantation and survival of tumour cells. Melanoma-derived exosomes, with their enriched miRNA cargo (e.g., miR-155, miR-210, miR-214), are primarily involved in this intercellular communication (for review: [35][36]). For instance, exosomes recruited into sentinel lymph nodes promote the upregulation of proteases that degrade the ECM and enhance the expression of pro-angiogenic factors (TNFα, VEGF, etc.) to facilitate the recruitment, trapping and growth of malignant melanocytes within the metastatic niche [37][38]. They are also capable of priming bone-marrow-derived cells (BMDCs) to achieve a vasculogenic, metastatic phenotype, and facilitate their recruitment to metastatic sites where they will contribute to the establishment of a suitable microenvironment for trapping circulating melanoma cells [39].

    3. LncRNAs Modulate Melanoma Invasion and Metastasis

    Despite the emerging interest and growing evidence of the contribution of lncRNAs in cancer [40][41], little is known about the impact of deregulated expression of lncRNAs in the invasion and metastasis of MM [2][42] (Figure 1).

    The first lncRNA characterized was SPRY4-IT1, a transcript derived from an intron of the SPRY4 gene, whose expression is increased in melanoma [43]. It was identified as a regulator of several processes, as suppression of SPRY4-IT1 resulted in abnormal cell growth, differentiation and apoptosis, as well as decreased invasion capacity of melanoma cell lines [43][44]. Although the molecular mechanisms that interfere with the invasion of MM are not clear, a study concerning non-small cell lung carcinoma revealed a possible role in the activation of EMT by modulating both E-cadherin and vimentin expression, leading to cell proliferation and metastasis [45]. Interestingly, another lncRNA with a potential role in regulating EMT was discovered by Siena et al., while exploring the lncRNA gene expression patterns across melanocytes, primary and metastatic melanoma cells. They found a significant upregulation of ZEB1 antisense RNA 1 (ZEB1-AS1) in melanoma cells. Data analysis from TCGA confirmed the increased expression of ZEB1-AS1 in metastatic melanoma and its association with hotspot mutations in BRAF and RAS family genes. Additionally, enrichment analysis correlated ZEB1-AS1 with the gene expression of zinc finger E-box binding homeobox 1 (ZEB1), an essential EMT marker, suggesting a possible role in melanoma invasiveness and phenotype switching [46].

    TCGA data analysis also confirmed the clinical relevance of SRA-like non-coding RNA1 (SLNCR1), a lncRNA whose highly conserved sequence is strikingly similar to that of lncRNA steroid receptor RNA activator 1 (SRA1), and whose increased expression is associated with shorter overall survival in melanoma patients. Functional and mechanistic studies revealed SLNCR1 promotes melanoma invasion through upregulation of MMP9 (involved in ECM degradation) in cooperation with the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) [47].

    A screen of differentially expressed lncRNAs in BRAF V600E mutated melanoma cells has led to the identification of BRAF-activated non-coding RNA (BANCR) as a putative regulator of cell proliferation and motility. Overexpression of BANCR in MM promotes the activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) components of the MAPK pathway, while its knockdown affects the migratory capacity of tumour cells [48][49]. A positive feedback mechanism with the V600E mutation is thought to induce BANCR overexpression, subsequent upregulation of chemokines and increase in melanoma cell motility [49][50].

    The oncogenic activity of antisense non-coding RNA in the INK4A locus (ANRIL) in melanoma revolves around the regulation of its encoding locus that also harbours the tumour suppressor genes INK4A and INK4B. In cutaneous melanoma tissue samples and cell lines, strong levels of ANRIL negatively modulated the expression of CDKN2A/2B proteins [51][52]. In addition, knockdown experiments managed to restore INK4A and INK4B expression, at the same time suppressing in vitro colony formation and migration [53]. Meanwhile, the encoding gene of survival associated mitochondrial melanoma-specific oncogenic lncRNA (SAMMSON) also harbours the melanoma-specific oncogene MITF and it was demonstrated that SAMMSON is frequently co-amplified with it [54]. As shown by functional assays, knockdown of this lineage-specific lncRNA drastically affects cell proliferation and viability, even sensitizing melanoma to MAPK-targeted drugs. Mechanistically, SAMMSON interacts with mitochondrial protein p32, a critical regulator of tumour metabolism [54].

    Although the molecular mechanisms are currently unknown, HOX transcript antisense intergenic RNA (HOTAIR) is perceived as a regulator of melanoma invasion and metastatic progression, considering it was found particularly enriched in lymph-node metastases compared to primary lesions and knockdown of HOTAIR in vitro suppressed melanoma cell motility and invasion [55]. In a similar manner, a significantly higher expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and urothelial carcinoma associated 1 (UCA1) was observed in advanced stages of MM, than in early stages, suggesting the potential role of MALAT1 and UCA1 in melanoma invasion and metastasis [56]. Moreover, a subsequent mechanistic study demonstrated that UCA1 can interact with miR-507 and promote the inhibition of FOXM1 expression, leading to increased invasiveness and clonogenic potential of melanoma cells [57].

    Recently, the upregulated expression of X-inactive-specific transcript (XIST) in MM tissues and resistant cell lines was reported, with XIST being proposed as a crucial regulator in melanoma progression [58]. Through the use of bioinformatics, XIST was revealed to act as a molecular sponge for miR-21, which targets PI3KR1, a regulatory subunit of PI3K. Functional studies concerning XIST repressed PI3KR1 and AKT expression, leading to inhibition of melanoma cell proliferation and migration, at the same time increasing sensitivity to oxaliplatin [58].

    Lastly, lncRNAs can also act as tumour suppressors in melanoma, however only growth arrest-specific transcript 5 (GAS5) and maternally expressed gene 3 (MEG3) have been reported so far [59][60]. Lentiviral-mediated overexpression of GAS5 diminished the expression of MMP2, a specific protein involved in ECM degradation, and reduced the migratory and invasive capacity of human MM cells [61]. Interestingly, GAS5 seems to repress melanoma tumorigenesis via miR-137, while MEG3 inhibits tumour growth and metastasis by modulating miR-21/E-cadherin axis [60][62].

    4. Non-Coding RNAs in Drug Resistance

    Continuous treatment with target-based therapies remains unsuccessful and leads to melanoma relapse due to acquisition of drug resistance. Even though BRAF monotherapy (BRAFi), or its combination with MEK inhibitors (MAPKi) instigate a profound regression in patients with BRAF-mutated metastatic melanoma, their effect is only temporary [63][64]. The same issue arises with the use of immunotherapy [65]. The role of ncRNAs in the development of melanoma drug resistance has been questioned and while the contribution of miRNAs is well studied [10], the role of lncRNAs in such resistance is still largely unknown.

    4.1. Resistance to BRAF or MAPK Inhibitors 

    The deregulated expression of several miRNAs, namely upregulation of onco-miRNAs and downregulation of oncosuppressors, has been shown to contribute to the acquisition of drug resistance to BRAFi or MAPKi-based therapy in melanoma (Table 1). For example, overexpression of miR-31a, miR-100 and miR-125b stimulates tumour cell proliferation and apoptosis escape, decreasing drug sensitivity in patients treated with vemurafenib. Their expression is associated with the chemokine monocyte chemoattractant protein-1 (CCL2), which promotes melanoma progression in BRAFi-resistant cells [66]. Inhibition of miR-125a leads to the partial drug resensitization of melanoma in a subset of BRAFi-resistant cell lines [67]. Also, it has been concluded that miR-204 and miR-211 facilitate the emergence of resistance to vemurafenib, as higher expression levels were found in resistant melanoma cells compared to their drug-naïve counterparts [68][69]. On top of that, low levels of miR-579-3p can affect not only the BRAF/MAPK signalling pathway, but also the MDM2/p53 pathway, causing uncontrolled cell proliferation and migration, coupled with inhibition of apoptosis, thus contributing to the development of MAPKi resistance [70]. Of note, overexpression of miR-579-3p was able to prevent colony formation in cells exposed to vemurafenib and impair the growth of BRAFi-resistant melanoma cells in combination with the MEK inhibitor (MEKi) trametinib [70].

    Recently, Sanlorenzo et al. identified MIRAT (MAPK Inhibitor Resistance Associated Transcript), a novel cytoplasmic lncRNA, which is significantly overexpressed in melanoma cells with acquired resistance to MAPKi, and modulates MAPK signalling by binding to the MEK scaffold protein IQGAP1 [71]. Knockdown of SAMMSON was shown to sensitize melanoma to MAPK-targeting therapeutics as well, but its underlying mechanism is unknown [54]. Meanwhile, Joung et al. performed a genome-scale activation screen and found EMICERI (EQTN MOB3B IFNK C9orf72 enhancer RNA I), a novel lncRNA that confers resistance to vemurafenib through upregulation of MOB3B and subsequent activation of the Hippo signalling pathway [72].

    Table 1. Non-coding RNAs that contribute to resistance or sensitivity to targeted therapy with BRAF or MAPK inhibitors.

    Induced Effect

    Overexpression of ncRNAs

    Target(s)

    Drugs

    References

    Drug resistance

    miR-34a, miR-100 and miR-125b

    CCL-2

    Vemurafenib

    [66]

    miR-125a

    BAK1, MLK3

    [67]

    miR-204 and miR-211

    NUAK1/ARK5, IGFBP5, TGF-bRII, Slug, CHD5

    [68][69]

    miR-514a

    NF1

    [73]

    MIRAT

    IQGAP1/MAPK signalling

    Trametinib (MEKi) and PLX4720 (BRAFi)

    [71]

    SAMMSON

    p32 mitochondrial protein

    Vemurafenib and pimasertib (MEKi)

    [54]

    EMICERI

    MOB3B/LATS1/Hippo signalling axis

    Vemurafenib

    [72]

     

     

     

     

     

    Drug sensitivity

    miR-7

    EGFR/IGF-1R/CRAF

    Vemurafenib

    [74]

    miR-32

    MCL-1

    [75]

    miR-200c

    Bmi-1

    Vemurafenib or analog PLX4720

    [76]

    miR-579-3p

    BRAF, MDM2

    Vemurafenib and trametinib

    [70]

    4.2. Resistance to Immunotherapy

    To date, results regarding the effect of ncRNAs on melanoma response to immune checkpoint blockers are sparse [77][78]. In order for studies to gain momentum in this area, some suggested the use of single-cell RNAseq approach to distinguish between the expression of cancer cells and that of the immune components [79]. Nevertheless, some miRNAs have been recognised as being involved in the conversion of monocytes into immunosuppressive MDSCs (let-7e, miR-99b, miR-100, miR-125a, miR-125b, miR-146a, miR-146b, miR-155) [80], while some interfere with PD-1 (miR-28) or PD-L1 (miR-17-5p) at a post-transcriptional level [81][82], facilitating resistance to immunotherapy (Table 2). LncRNAs have also been identified as potential modulators of myeloid cell differentiation towards an immunosuppressive phenotype (olfr29-ps1, lnc-chop) [83][84], however it seems that one particular polycistronic lncRNA, namely melanoma-overexpressed antigen (MELOE), can unexpectedly improve antigen presentation after being translated into short polypeptides, potentially increasing melanoma immunotherapy efficiency [85] (Table 2). 

    Table 2. Non-coding RNAs that interfere with immunotherapy in a negative or positive manner.

    Name of ncRNAs

    Function

    Immune Response

    References

    miR-28

    Blocks immune checkpoint

    Silences PD-1 by binding to its 3′UTR region

    Negative

    [81]

    miR-17-5p

    Blocks immune checkpoint ligand

    Binds to PD-L1 and contributes to melanoma resistance

    Negative

    [82]

    Let-7e, miR-99b, miR-100, miR-125a, miR-125b, miR-146a, miR-146b, miR-155

    Control of MDSC

    Favours myeloid cell differentiation and polarization towards an immunosuppressive phenotype

    Negative

    [80]

    Olfr29-ps1

    Control of MDSC

    Promotes MDSCs’ differentiation and function via de m6A-modified Olfr29-ps1/miR-214-3p/MyD88 regulatory network

    Negative

    [83]

    Lnc-chop

    Control of MDSC

    Promotes the differentiation and function of MDSCs

    Negative

    [84]

    MELOE

    Antigen presentation

    Produces immunogenic antigens (MELOE-1 and -2) that are recognized by cytotoxic T cells

    Positive

    [85]

    5. Clinical Applications of Non-Coding RNA Molecules in Melanoma Management 

    The clinical utilities and implications of ncRNAs in melanoma management are not fully established and future investigations are needed to clarify this aspect [2][79], however some promising results that recommend miRNAs and lncRNAs as tools for diagnosing, monitoring and treating cutaneous melanoma are presented in the following sections.

    5.1. Non-Coding RNAs as Circulating Biomarkers for Cutaneous Melanoma

    Even though lots of therapeutic progress has been made throughout the past years in finding alternative options for melanoma prognosis, it still remains challenging to find minimal invasive methods to follow up cancer progression and metastasis. Thus, liquid biopsies rise as a useful tool for the detection of circulating cancer biomarkers, among them ncRNAs [86][87][88].

    Up to date lots of effort and work has been put in identifying a landscape of miRNAs as circulating biomarkers for melanoma diagnosis and prognosis (Table 3). Among the first to address this matter, Leidinger et al. have performed high throughput methods to validate a spectrum of 16-miRNAs which could be identified only in melanoma positive patients [89]. Several studies have indicated an increased level of multiple miRNAs, such as miR-19a, miR-149 and miR-126, in the plasma of metastatic melanoma patients as compared to healthy controls, suggesting these molecules to be actively involved in melanoma progression [90][91][92]. Interestingly, the co-detection of miR-185 and miR-1246 in liquid samples could offer an accurate identification of patients with metastatic melanoma, which could allow an early cancer diagnosis [93]. Additionally, Van Laar et al. have proposed a 38-miRNA signature (MEL38) in order to designate melanoma from normal plasma samples and an 18-miRNA signature (MEL18) which has the means to differentiate non-metastatic (stage I/II) and metastatic (stage III/IV) melanoma subjects [94]. A study done by Li et al. has detected miR-221 in serums samples from cutaneous MM patients, making this molecule a potential biomarker for melanoma evolution [95]. Another 4 miRNAs molecules (miR-30d, miR-15b, miR-150 and miR-425) have been proposed as potential biomarkers in order to discriminate between low and high-risk cases of recurrence [96]. Furthermore, Stark et al. indicated a 7-miRNA panel (MELmiR-7) including miR-16, miR-509-5p, miR-4706 and miR-211-5p which could be successfully implemented in the prediction of melanoma growth and recurrence, thus having a significant impact on melanoma prognosis and diagnosis [97].

    In a similar manner, lncRNA molecules have also been identified as potential circulating biomarkers for melanoma (Table 3). In this context, it has been demonstrated that plasma lncRNA SPRY4-IT is significantly higher in tumour samples as compared to healthy controls [98]. Furthermore, Tang et al. observed the overexpression of HOTAIR in melanoma serum samples in comparison to non-cancer probes [55], while Cantile et al. found a significantly higher expression of HOTAIR in serum samples taken from patients with advanced melanoma [99]. These promising reports support the fact that HOTAIR has strong implications in the carcinogenesis of melanoma, making it a potential prognostic and diagnostic marker for MM.

    Table 3. Promising results that recommend non-coding RNAs as circulating biomarkers for potential clinical applications.

    Potential Role

    Non-Coding RNAs

    Sample Type

    References

    Circulating biomarkers for prognosis and diagnosis

    16 miRNA panel (among them miR-30d and miR-17)

    Blood

    [89]

    miR-19a

    miR-126

    miR-149

    Plasma

    [90][91][92]

    miR-185

    miR-1246

    [93]

    18 miRNA panel (among them miR-199b-5p and let-7e)

    [94]

    miR-221

    Serum

    [95]

    miR-15b

    miR-30d

    miR-150

    miR-425

    [96]

    7 miRNA panel

    (among them miR-16, miR-211-5p, miR-4706 and miR-509)

    [97]

    SPRY4-IT

    Plasma

    [98]

    HOTAIR

    Serum

    [55][99]

    5.2. Non-Coding RNAs as Targets for Promising Therapeutic Strategies

    The potential of targeting ncRNAs to develop novel anticancer therapeutic strategies or to increase the efficacy of already existing ones has been pointed out in several studies [79][100][101].

    A number of ways in which miRNAs could be used have already been analysed, among which (1) synthetic miRNA mimetic agents, that could replace lost miRNA, (2) small-molecule inhibitors of miRNA, used for suppressing miRNA biogenesis or interaction with its target, (3) anti-miRNA oligomers, which are competitive inhibitors of miRNAs, leading to an upregulation of the target mRNA, or even (4) directly targeting miRNAs in the course of their transport within the tumour milieu or to other sites [102]. For example, miR-200c has been described as a potential therapeutic target for overcoming resistance to BRAFi therapy. In BRAFi-resistant cell lines and more importantly in clinical samples, low levels of miR-200c are correlated with acquired resistance. Restoration of miR-200c expression or knockdown of its molecular target favours the effect of inhibitory drugs and impairs the establishment of resistance [76] (Table 4). However, the use of miRNAs in therapy is hampered by their poor intracellular uptake, as well as rapid degradation in biological fluids. Strategies to deliver tumour-suppressive miRNAs or interfere with tumour-promoting miRNAs are still under development [103]. For instance, Fattore et al. developed lipid nanoparticles to encapsulate miR-204-5p and miR-199b-5p, either individually or in combination, and tested them on in vitro drug resistant models. Their results showed that these lipid nanoparticles loaded with oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affecting key signalling cascades involved in cell survival, in addition to positively influencing the efficacy of BRAF and MEK inhibitory drugs [104] (Table 4).

    The involvement of lncRNAs in melanoma has promising therapeutic implications and selective knockdown of specific lncRNAs could lead to the development of reliable therapeutic strategies (Table 4). Out of the current methods available for studying lncRNAs, small interference RNA (siRNA)-dependent knockdown is used the most [105]. For instance, siRNA-mediated knockdown of SPRY4-IT1 in melanoma cell lines prevented tumour cell growth and limited invasion [43][44]. Similar results were obtained for knockdown of HOTAIR and UCA1, including inhibition of cell motility and invasive capacity [55][57]. MALAT1 knockdown was followed by a decrease in melanoma cell migration [56], whereas colony formation and metastatic ability of cancer cells were diminished in the absence of ANRIL [53]. Knockdown of SLNCR1 decreased the invasiveness of melanoma cells, although cell proliferation and motility were not affected [47]. Administration of GAS5, a tumour suppressor lncRNA, to nude mice inhibited melanoma growth, however further studies focused on the therapeutic value of GAS5 are needed [59].

    The combination of a SAMMSON-specific antisense oligonucleotide with the BRAFi dabrafenib exerted a synergistic anti-tumour effect and induced apoptosis in a patient derived tumour xenograft preclinical model, whereas dabrafenib could only restrain melanoma growth (Table 4). Moreover, no significant difference was found between the toxicity levels of administrating SAMMSON-specific antisense oligonucleotide with BRAF inhibitors in comparison to administration of BRAF or MEK inhibitors alone [54].

    Table 4. Promising results that recommend non-coding RNAs as therapeutic targets for potential clinical applications.

    Potential Role

    Functional Studies

    Research Model

    Therapeutic Effect(s)

    References

    Targets for promising therapeutic strategies

    Lentiviral overexpression of miR-200c

    BRAFi-resistant cell lines

    Restores sensitivity to BRAFi therapies

    [76]

    Lipid nanoparticles loaded with miR-204-5p and/or miR-199b-5p

    In vitro drug resistant models

    Impair melanoma cell proliferation and viability

    Positively influence the effect of MAPKi

    [104]

    siRNA-mediated knockdown of SPRY4-IT1

    Malignant melanoma cell lines

    Prevents tumour cell growth and limits invasion

    [43][44]

    siRNA-mediated knockdown of HOTAIR

    Inhibits cell motility and decreases invasion

    [55]

    siRNA-mediated knockdown of UCA1

    Inhibits cell proliferation and invasion

    Induces cell cycle arrest

    [57]

    siRNA-mediated knockdown of MALAT1

    Impairs melanoma cell migration

    [56]

    siRNA-mediated knockdown of ANRIL

    Diminishes colony formation and metastatic ability

    [43]

    siRNA-mediated knockdown of SLNCR1

    Decreases invasiveness of melanoma cells

    [47]

    Lentiviral overexpression of GAS5

    In vitro and in vivo models

    Inhibits melanoma growth and cell migration

    [59]

    SAMMSON-specific antisense oligonucleotide

    Patient- derived xenograft

    Induces apoptosis

    Exerts a synergistic anti-tumour effect with dabrafenib

    [54]

    6. Conclusions

    Research concerning the molecular landscape of malignant melanoma has brought impressive results in terms of patients’ overall survival in metastatic disease, due to its contribution to the development of targeted-based drugs and immunotherapy. Nevertheless, acquired resistance to therapy still remains a challenge, reflecting upon the poor prognosis of a significant number of patients. In this regard, therapeutic strategies aimed to modulate ncRNAs in combination with targeted agents and/or immunotherapy may represent a more efficient solution, considering that miRNAs and lncRNAs are not only involved in melanoma invasion and metastasis, but also facilitate resistance against currently available molecular therapeutic approaches. Strategies to deliver tumour-suppressive ncRNAs or interfere with tumour-promoting ncRNAs are searched for and future directions for the development of innovative treatment modalities include the use of intelligent nanocarriers loaded with ncRNAs for selective gene therapy. Although miRNAs and lncRNAs seem very promising biomarkers, their translation into the clinical area requires further studies and future investigative clinical trials.

    The entry is from 10.3390/cancers12113378

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