MiR-21: an environmental driver of malignant melanoma?

MiR-21 is a key oncogene, which is highly expressed in most cancers [18, 19]. Critical targets of miR-21 are mRNAs of tumor suppressor proteins, checkpoint regulators of cell cycle control, and intrinsic and extrinsic pathways of cellular apoptosis [20]. MiR-21 inhibits mRNA expression of crucial tumor suppressor proteins such as PTEN (phosphatase and tensin homolog) [21, 22], Sprouty1 and Sprouty2 [23‐25], and PDCD4 (programmed cell death protein 4) [26‐29]. MiR-21 is a negative regulator of p53 signaling [30] and stimulates the expression of the cell cycle promoter cyclin D1 [31]. MiR-21 induces tumor angiogenesis through targeting PTEN, leading to activated AKT and ERK1/2 signaling, thereby enhancing hypoxia-inducible factor 1α (HIF1α) and the expression of the vascular endothelial growth factor (VEGF) [32]. HIF1α is a crucial downstream target of miR-21 in regulating tumor angiogenesis [32‐34]. Overexpression of HIF1α and HIF2α is linked to VEGF expression and poorer prognosis in malignant melanomas [35, 36] (Figure 1).

APAF1, apoptotic protease activating factor-1, which is the molecular core of the apoptosome, has been identified as a miR-21 target [37]. Metastatic melanomas often lose APAF1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis [38, 39]. There is an inverse correlation between APAF1 expression and melanoma progression [40, 41]. In fact, a significant difference in APAF1 mRNA expression between melanomas of Breslow thickness <1 and >4 mm has been detected [41]. Thus, there is good reason to believe that increased miR-21 expression via suppression of APAF1 prevents apoptosis in melanoma. Further studies confirm the involvement of miR-21 in the pathogenesis, carcinogenesis, progression and metastasis of malignant melanoma [13, 14, 42‐45].

Mutations in the BRAF gene and less frequently such as PTEN, KIT, CDK4, p53, MDM2, cyclin D1, AKT3, PI3Kα, or NRAS are involved in melanoma progression [10‐12]. Important negative regulators of NRAS/BRAF/MEK/ERK- and the PI3K/AKT/mTORC1 signaling pathways are targets of miR-21 (Figure 2). Sprouty proteins are negative master regulators of RAF-RAS signaling [46]. MiR-21-mediated inhibition of Sprouty may thus promote mutated BRAF/NRAS signaling of melanoma cells. MiR-21 augments PI3K/AKT/mTORC1 signaling in cancer cells at various levels of the pathway [47, 48]. Recent evidence suggests that miR-21-mediated gene regulation interconnects with the AKT pathway [48]. In PC3 cells, miR-21 expression resulted in a dramatic increase in basal levels of the PI3K subunit p85 [49]. PI3K signaling is required for TGFβ-induced epithelial-mesenchymal (EMT)-like transition in human melanoma cells [50].

PTEN, which suppresses PI3K/AKT signaling, is often mutated in melanomas [12, 51]. Inactive mutated PTEN leads to overactivation of AKT, which inactivates FOXO tumor suppressor proteins [52].

Recent evidence shows that miR-21 not only targets PTEN, but also FOXO1 mRNA [53, 54]. Decreased FOXO activity has been associated with the malignant phenotype of melanoma cells [55, 56]. FOXO3a stimulates apoptosis by negatively targeting miR-21 [57]. MiR-21 suppresses the translation of pro-apoptotic FAS ligand (FASLG) [57]. It is noteworthy to mention that FoxO3a is a transcription factor promoting FOXO1 upregulation [58]. Thus, there exists a transcriptional and posttranslational regulatory network between FOXO3a, FOXO1 and miR-21 expression. Nuclear activity of FOXO transcription factors depends on PI3K/AKT-mediated phosphorylation of FOXO proteins [59]. As the miR-21 target PTEN counteracts the activity of PI3K, miR-21 has an upstream influence on nuclear FOXO-mediated transcription such as the expression of FOXO-dependent target genes like FASLG, cyclin D1, p21, superoxide dismutase and catalase involved in the regulation of apoptosis, cell cycle progression and defense against reactive oxygen species (ROS) [60]. Taken together, miR-21 enhances upstream and downstream PI3K/AKT signaling and reduces FOXO activities.

PDCD4 is a negative regulator of translation and acts as a tumor suppressor [61]. Notably, mTORC1 stimulates mRNA translation and protein synthesis via S6K1-mediated phosphorylation of PDCD4 (Figure 2) [62]. PDCD4 is suppressed in ~25% of human cell lines that are established from advanced melanoma lesions [63]. PDCD4 is a target of miR-21 [26‐29]. Thus, overexpression miR-21 is involved in EMT by targeting PDCD4 and PTEN [64‐67].

BTG2 (B-cell translocation gene 2) encodes an antiproliferative protein involved in the regulation of the G1/S transition of the cell cycle [68]. The tumor suppressor BTG2 is relevant to cell cycle control and cellular response to DNA damage [69]. BTG2 acts as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic RAS [70]. BTG2 is a known target of miR-21 [71]. Knockdown of miR-21 in B16 melanoma cells increases BTG2 levels [42], indicating that BTG2 is a miR-21 target in melanoma cells.

Another recently identified target of miR-21 is the insulin-like growth factor binding protein 3 (IGFBP3) [72]. Along with having a number of IGF-independent effects on cell growth and survival, IGFBP3 is known to modulate the activity of insulin-like growth factors (IGFs) [73]. In glioblastoma, miR-21-mediated downregulation of IGFBP3 expression promotes tumorigenesis [72]. Recent evidence underscores that IGFBP3 exerts a specific inhibitory effect on melanoma growth and dissemination [74].

Recently, the tumor suppressor FBXO11 has been identified as a novel miR-21 target [75]. FBXO11 is a component of the SKP1-CUL1-F-box ubiquitin ligase complex that targets proteins for ubiquitination and proteasomal degradation [76], a regulatory mechanism that plays a crucial role in the maintenance of genome stability [77]. FBXO11 promotes apoptosis by mediating the degradation of oncogenic BCL6. Notably, FBXO11 acts as a tumor suppressor in melanoma and has been shown to regulate apoptosis of B10BR mouse melanocytes [78].

Another recently detected miR-21 target is the tissue inhibitor of metalloproteinases 3 (TIMP3) [79]. Increased miR-21 expression enhances the invasive potential of melanoma cell lines through TIMP3 inhibition by (1) increasing matrix metalloproteinase activity [80], (2) by stimulating angiogenesis via TIMP3-mediated blockade of VEGF binding to VEGFR2 [80], and (3) by attenuating TIMP3-mediated apoptosis. Notably, TIMP3 exhibits potent antitumor activity in an animal model of melanoma [81] and induces apoptosis in melanoma cells [82]. Adenovirally expressed TIMP3 stabilzes tumor necrosis factor receptor-1 (TNF-R1), FAS, and TNF-related apoptosis, inducing ligand receptor-1 (TRAIL-R1) on melanoma cell surface, sensitizing these cells to apoptosis induced by TNF-α, anti-FAS-antibody and TRAIL [83]. Thus, the miR-21 target TIMP3 promotes apoptosis in melanoma cells by stabilizing three distinct death receptors and activating their apoptotic signaling cascade through caspase-8 (Figure 1).

Integrin-β4 (ITGβ4) is a novel miR-21 target gene and plays a role in the regulation of EMT, as it is remarkably derepressed after transient miR-21 silencing and downregulated after miR-21 overexpression. MiR-21-dependent changes of ITGβ4 expression significantly affect cell migration properties of colon cancer cells [84].

Expression of the L1 cell adhesion molecule (L1CAM) frequently occurs in human cancers and is associated with poor prognosis in cancers. It has recently been demonstrated that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways [85]. Notably, miR-21-3p has been identified as a positive regulator of L1CAM expression [86].

Overexpression of tropomyosin 1 (TPM1) in MCF-7 cells suppresses anchorage-independent growth, whereas overexpression of miR-21 increases tumor growth. Zhu et al. [87] concluded that miR-21 acts as an oncogene by suppressing TPM1. Indeed, increased expression of TPM1 has been associated with decreased invasive and motile activities of melanoma cells [88, 89].

The DNA mismatch repair (MMR) protein MSH2 is an important tumor suppressor and crucial caretaker of the MMR, including transcription-coupled repair [90], homologous recombination [91], base excision repair [92], and plays an important role in mutation avoidance and microsatellite stability [93]. MSH2 is involved in the repair of UVA-induced oxidative DNA damage by base excision repair [94]. MSH2 gene mutations are present in the radial growth-phase of cutaneous malignant melanoma cell lines and can be further induced by UV-B [95]. Reduced or defective expression of MSH2 has been associated with high genomic instability, poor melanoma prognosis, and metastasis [95, 96]. Reduced expression or function of MSH2 is either a result of mutation-derived dysfunction of MSH2 [94‐97] or miR-21-mediated downregulation of MSH2 mRNA [98] (Figure 1).

Telomerase is reactivated in most cancers and there is accumulating evidence that this is a driver event in malignant melanoma [99‐102]. hTERT is the catalytic subunit of telomerase, which regulates telomerase activity. Wang et al. [103] demonstrated in glioblastoma cells that enforced miR-21 expression increases hTERT expression mediated by STAT3, thereby promoting glioblastoma cell growth, whereas reduction of miR-21 represses hTERT expression in a STAT3-dependent fashion.

The development and progression of melanoma can be attributed to independent or combined genetic and epigenetic events [104]. The MIR21 promoter region includes binding sites for activating protein 1 (AP-1) and signal transducer and activator of transcription 3 (STAT3) [19]. The AP-1 transcription factor c-Jun is highly expressed in melanoma cells [105]. AP-1/c-Jun activation results in enhanced expression of miR-21 [106‐109]. Primary human melanocytes genetically modified to ectopically express BRAF(V600E) or NRAS (G12D) induce c-Jun expression [108‐110] (Figure 2). RAS oncogenes are well-characterized inducers of AP-1 activity [109]. Indeed, oncogenic RAS induction in thyroid cells increases the expression of miR-21 [111]. Furthermore, an autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation [112].

Melanoma cells exhibit reduced expression of miR-125b, which is a negative regulator of c-Jun [113]. Thus, miR-125b/c-Jun/miR-21 signaling may represent a further pathway linked to miR-21-dependent melanomagenesis.

STAT3 is constitutively activated in a majority of human melanoma cell lines and tumor specimens [114, 115]. STAT3 activity is required for melanomagenesis and increases tumor invasiveness [116]. Noteworthy, STAT3 stimulates the expression of miR-21 [30, 40, 117]. In fact, STAT3/miR-21-signaling promotes proliferation and metastasis of B16 melanoma cells [42]. Inhibition of STAT3 is regarded as a potential therapeutic approach to target melanoma [118].

Approximately 50% of melanomas depend on mutant BRAF for proliferation, metastasis and survival [119]. Activation of STAT3 serine-727 and tyrosine-705 phosphorylations is promoted by BRAF(V600E) activity [118], whereas MEK inhibition decreases STAT3 phosphorylation in NRAS-mutant melanoma [119]. Furthermore, increased STAT3 signaling has been reported in primary oncogenic driver mutations of KIT, ALK, ROS1, RET and NTRK1 [12]. In addition, RAS-induced expression of miR-21 is mediated through STAT3 signaling [20]. Taken together, common driver mutations of malignant melanoma via AP-1 and STAT3 activate miR-21 signal transduction.

MiR-182 is upregulated in melanoma cell lines [8, 120]. Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor (MITF) [120]. It has recently been demonstrated that miR-182 is upregulated in melanoma cell lines after epigenetic modulation with the demethylating agents 5-aza-2′-deoxycytidine and trichostatin A [121]. It is concerning that miR-182 downregulates the expression of FOXO3 [121], which is a critical repressor of MIR21 [57]. Thus, enhanced miR-182 signaling due to oncogenic mutations or epigenetic upregulation may synergistically augment miR-21 expression in melanoma. The intertwined connection between epigenetics and miRs such as miR-182 modulate the activity of the epigenetic machinery that plays a role in cancer development [122, 123].

Dermal fibroplasia appears to be related to the degree of atypia in dysplastic nevi [124]. Periadnexal fibrosis has been observed in melanoma in situ [125]. Thus, subepidermal fibroplasia may be a co-feature of melanomagenesis. Recent evidence links skin fibrosis to miR signaling [126]. MiR-21 promotes fibrogenic EMT of epicardial mesothelial cells involving PDCD4 and Sprouty-1 [127]. MiR-21 has been identified as central regulator of fibrosis [128].

Cancer-associated fibrosis plays an important role for the tumor stroma that supports cancer growth and invasion. Inhibition of miR-21 reduces liver fibrosis and prevents the development of hepatocellular carcinoma [129]. A paracrine signaling network involving PDGF-CC and PDGF receptor-α in a mouse model of malignant melanoma accelerates tumor growth through recruitment and activation of different subsets of cancer-associated fibroblasts (CAFs) [130]. TGF-β1 is a pivotal signal that promotes the generation of CAFs. Li et al. [131] demonstrated that miR-21 targets SMAD7, the upstream inhibitor of TGF-β1 signaling. MiR-21 binds to the 3′ UTR of SMAD7 mRNA and inhibits its translation [131]. Overexpression of miR-21 or the depletion of SMAD7 promoted CAF formation [131]. These observations point to an important contribution of miR-21 in shaping melanoma’s stromal microenvironment. Increased perilesional fibrosis may thus reflect the histologic result of enhanced miR-21 expression during the transition from a benign melanocytic lesion to malignant melanoma [14].

MiR-21 is a major miR component of human plasma that increases in association with age, inflammation, cardiovascular disease, and obesity [189‐191]. STAT3 activation of miR-21 has been proposed as a mechanistic link between inflammation and cancer development [192]. Inflammasome activation and increased IL-1β signaling has recently been proposed to play a potential role in driving melanomagenesis [193]. Serum IL-1β is significantly increased in advanced melanoma patients [194]. Remarkably, it has been demonstrated in human aortic epithelial cells that IL-1β treatment induces a three to fourfold response of miR-21-3p expression compared with control treatment [195]. This can be explained by IL-1β-induced IL-6/STAT3 signaling [196].

Inflammation is a newly recognised hallmark of cancer that substantially contributes to the development and progression of malignancies [197]. Increasing evidence underlines the role that local immune response and systemic inflammation have in the progression of tumors and survival of cancer patients. It has recently been proposed that miR-21 plays a key role in the induction and resolution of inflammatory responses [198] and in the regulation of immune homeostasis [196]. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), which belongs to the TNF-α-induced protein-8 family, is a negative regulator of innate and adaptive immunity and is a direct target of miR-21 [199, 200]. TIPE2 negatively regulates inflammation by switching arginine metabolism from nitric oxide synthase to arginase, which converts arginine to ornithine and urea [201]. Melanomas are auxotrophic for arginine due to the reduced expression of argininosuccinate synthetase-1 (ASS1), which is the rate-limiting enzyme for arginine biosynthesis [202]. Furthermore, miR-21-regulated TIPE2 controls innate immunity to RNA by targeting the PI3K-RAC pathway [203]. TIPE2 is able to suppress TNF-α-induced hepatocellular carcinoma metastasis by inhibiting ERK1/2 and NF-κB activation [204]. Downregulated TIPE2 is associated with increased cell proliferation and poor prognosis in non-small lung cancer [205]. Furthermore, TIPE2 provides a molecular bridge from inflammation to cancer by targeting the RAS signaling pathway [206, 207]. TIPE2 binds the RAS-interacting domain of the RALGDS (RAL guanine nucleotide dissociation stimulator) family of proteins, which are essential effectors of activated RAS. This binding prevents RAS from forming an active complex, thereby inhibiting the activation of the downstream signaling molecules RAL and AKT [207]. Thus, miR-21 reduces TIPE2-mediated suppression of RAS-induced tumorigenesis, which may have a potential impact on RAS-driven melanomagenesis.

Smoking and nicotine exposure upregulates the expression of miR-21 [67, 230]. Chronic arsenic exposure acts as a co-carcinogen in melanoma [231]. Arsenic exposure induces the expression of miR-21 [65, 232‐235]. A recent study shows that miR-21 is involved in exosome-mediated intercellular communication between neoplastic and normal human bronchial epithelial (HBE) cells [236]. Exosomes derived from arsenite-transformed HBE cells stimulate proliferation of normal HBE cells, whereas exosomes from miR-21-depleted cells fail to stimulate proliferation. Collectively, these data support the concept that exosomal miR-21 is involved in cell–cell communication during carcinogenesis induced by environmental chemicals. Furthermore, air pollution and oxidative stress induce the expression of miR-21. It has been demonstrated that metal-rich particulate matter increases the expression of miR-21 in peripheral blood leukocytes [237]. Notably, miR-21 expression has been associated with exposure to diesel exhaust linked to increased plasma levels of 8-hydroxy-deoxyguanosine (8-OHdG) [238]. 8-OHdG is induced in DNA by oxidative stress and UV irradiation [239]. Indeed, melanoma patients with low expression of nuclear 8-OHdG have significantly longer survival times compared to those with high expression [240]. Thus, air pollution and urbanisation may affect melanomagenesis via environmental stressors that upregulate miR-21 expression (Table 1).