RTL1 promotes melanoma proliferation by regulating Wnt/β-catenin signalling

Cutaneous melanoma is a highly malignant and metastatic skin cancer with high mortality. However, its underlying mechanisms remain largely unclear. Here, we found that retrotransposon-like 1 (RTL1) is highly enriched in melanoma tissue, especially in early and horizontal growth tissues. Knockdown of RTL1 in melanoma cells resulted in cell proliferation suppression; cell cycle arrest at G1 phase; and down-regulation of E2F1, CYCLIN D1, cyclin-dependent kinase 6 (CDK6) and c-MYC. Moreover, overexpression of RTL1 in melanoma cells accelerated cell proliferation, promoted passage of the cell cycle beyond G1 phase, and increased the expression of cell cycle related genes. Mechanistically, we found that knockdown of RTL1 inhibited the Wnt/β-Catenin pathway by regulating the expression of genes specifically involved in β-CATENIN stabilization. Furthermore, the overexpression and knockdown of β-CATENIN rescued the effects of RTL1 on melanoma cell proliferation and the cell cycle. These findings were also confirmed via tumour xenografts in nude mice. Together, our results demonstrated that RTL1 promotes melanoma cell proliferation by regulating the Wnt/β-Catenin signalling pathway.


INTRODUCTION
Cutaneous melanoma is a highly aggressive skin cancer and is responsible for over 80% of all deaths among skin cancer patients. The mortality of melanoma increased nearly 165% worldwide from the 1950s to 2000 [1], and the incidence has increased continuously over the past decade [2][3][4]. Because malignant melanoma is not sensitive to chemotherapy or radiotherapy, both of which are deleterious to a patient's immune system [5][6][7], surgery is the most effective treatment for early melanoma with a 5-year survival rate of 89-96%. However, the time window for surgery is very narrow, and many patients miss the chance to undergo surgery, owing to metastasis. Recently, gene therapy and immunotherapy have emerged as potential therapeutic approaches for melanoma, although the underlying mechanisms remain to be elucidated.
Retrotransposon-like 1 (RTL1) is a Ty3/Gypsy transposon located in the conserved Dlk1-Dio3 imprinted region [8]. RTL1 plays an important role in maintaining the normal placental barrier, and it affects the growth of embryonic blood vessels when it is abnormally expressed, thus causing embryonic deformity or embryo deaths [9]. Interestingly, RTL1 has recently been reported to be associated with the development of several cancers. The methylation level of the RTL1 promoter is significantly www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 62), pp: 106026-106037 Research Paper lower in lung cancer tissues than in normal lung tissues. Further, the expression of microRNAs in the exon of RTL1 is increased [10], and RTL1 is highly expressed in mouse liver cancer induced by the Sleeping Beauty (SB) gene [11]. Because RTL1 is the only consistently altered gene detected in all SB-induced tumours with Dlk1-Dio3 integrations, it has been suggested that RTL1 activation is a driver of liver cancer [12]. Recently, it has been reported that the expression level of RTL1 was significantly increased in the squamous cell carcinoma of head and neck, but the function of RTL1 was not described [13]. Thus, little is known about whether RTL1 is involved in the development of melanoma.
In the present study, we observed that RTL1 was highly expressed in melanoma tissues, and was critically involved in the proliferation of melanoma cells in vitro and tumour growth in vivo. Further, we found that RTL1 activates the Wnt/β-Catenin signalling pathway by regulating the expression of β-Catenin stabilizing proteins, thus promoting cell cycle progression in melanoma cells.

RTL1 is highly expressed in human skin melanoma
To explore the role of RTL1 in melanoma, we examined the expression of RTL1 in the nevus and melanoma, and found that RTL1 was highly expressed in melanoma tissues. Quantitative real-time PCR (QPCR) analysis confirmed the significantly higher expression of RTL1 in melanoma tissues than in nevus tissues ( Figure 1A, 1B). Tissue array analysis was also used to detect the expression of RTL1 in normal skin, nevus, cutaneous squamous cell carcinoma, basal cell carcinoma, and melanoma (Table 1). These findings showed that RTL1 was more frequently upregulated in melanoma than in other tissues ( Figure 1C, 1D). The results of the positive rate in different melanoma phases showed that the expression of RTL1 in early and horizontal growth melanoma was higher than that in the vertical growth and metastasis states ( Figure 1E, 1F). These results indicated a significant role of RTL1 in early stages of melanoma.

RTL1 promotes the proliferation of melanoma cells
To examine the expression of RTL1 in melanoma cells, a keratinocyte line (HaCat) and two melanoma cell lines (A875 and A375) were used in the study. We found that both the protein and mRNA levels of RTL1 were significantly higher in melanoma cells than in HaCat cells (Figure 2A, 2B). To explore the exact function of RTL1 in melanoma, we generated lentiviral vectors containing the full-length cDNA and shRNAs targeting RTL1 to overexpress and knockdown RTL1, respectively. The efficiency was confirmed by QPCR and western blot ( Figure 2C, 2D).
A cell counting kit (CCK-8) and flow cytometry assays were used to investigate the role of RTL1 in melanoma. The results showed that knockdown of RTL1 inhibited the proliferation of melanoma cells and arrested the cell cycle transition from the G1 to the S phase, whereas overexpression of RTL1 promoted cell proliferation with cell cycle acceleration in the G1 phase. Furthermore, the cell cycle related genes were detected, and the expression levels of E2F1, CYCLIN D1, cyclin-dependent kinase 6 (CDK6) and c-MYC were up-regulated with RTL1 overexpression, whereas the knockdown of RTL1 down-regulated these genes ( Figure  2E-2J). Additionally, the protein expression of these genes was detected by western blot analysis ( Figure 2K

RTL1 activates the Wnt/β-Catenin pathway in melanoma cells
The Wnt/β-Catenin pathway has been reported to be aberrantly activated in melanoma cells. To further investigate the functional mechanism of RTL1 in regulating cell proliferation in melanoma, we observed the effects of RTL1 on the activation of the Wnt/β-Catenin pathway by using TOP/FOP Flash luciferase reporter gene analysis. The results showed that overexpression of RTL1 enhanced the activation of Wnt signalling, whereas knockdown of RTL1 had the opposite effects ( Figure  3A). However, the mRNA level of β-Catenin was not affected by modulating RTL1 expression. Furthermore, we observed the expression of β-Catenin and its stabilizing genes by QPCR, and we found that DOCK4, EPHB2, MACF1 and PLAUR were increased upon RTL1 overexpression and decreased with RTL1 knockdown ( Figure 3B). Using western blot analysis, we found that the protein level of β-CATENIN was elevated after RTL1 overexpression, and it was decreased in the RTL1knockdown cells ( Figure 3C). We have also performed the corresponding experiments in lung cancer cell line (A549) and liver cancer cell line (HepG2), and confirmed the effects of RTL on Wnt signaling and β-Catenin stabilizing genes (Supplementary Figure 2A and 2B). Furthermore, we observed that the expression of β-CATENIN was significantly higher in human melanoma tissue than in the nevus by immunohistochemistry analysis ( Figure 3D).

Figure 1: RTL1 is highly expressed in human skin melanoma. (A)
Immunohistochemical analysis of the expression of RTL1 in human melanoma and naive tissues; three tissues were examined; (B) The expression of RTL1 was analysed using RT-PCR, as detailed in the Methods, *** p<0.001; (C, D) Tissue array analysis expression levels of RTL1 in control human skin, nevus, melanoma, and other skin cancer. The expression of RTL1 was higher than that in other tissue; (E, F) The RTL1 staining positive rates in the early phase, horizontal growth, vertical growth, and metastasis states were compared, and the RTL1 positive rate was higher in the early phase, horizontal growth states.
The results of treatment with FH535, an inhibitor of Wnt signalling, showed that treatment of FH535 blocked the effects of RTL1 on cell proliferation and the cell cycle ( Figure 3E, 3F), thus further confirming the critical role of RTL1 on Wnt signalling. These results suggested that RTL1 affects cell proliferation through regulating the Wnt/ β-Catenin signalling pathway.

β-Catenin is a crucial target of RTL1 in melanoma
To further understand the role of RTL1 in the Wnt/β-Catenin pathway, we generated a lentiviral vector containing the β-Catenin cDNA and shRNAs targeting the β-Catenin sequence in vitro. Lentiviral particles containing β-Catenin cDNA or shRNAs were used to infect melanoma cells. The overexpression and knockdown efficiency of β-Catenin was confirmed by QPCR and western blot analysis ( Figure 4A, 4B). Using TOP/FOP Flash luciferase reporter gene analysis, we observed that in RTL1-overexpressing cells, knockdown of β-Catenin inhibited the activity of the Wnt pathway, whereas the overexpression of β-Catenin had a similar effect in RTL1knockdown cells ( Figure 4C). Moreover, overexpression of β-Catenin rescued the effect of RTL1 knockdown on cell proliferation, the cell cycle and the expression of cell cycle-related genes, whereas knockdown of β-Catenin reversed the promoting effect of RTL1 overexpression ( Figure 4D-4F). These results suggested that RTL1 affects the Wnt signalling pathway by modulating β-Catenin.

RTL1/β-Catenin engages in tumour growth in vivo
To validate the effect of RTL1/β-Catenin on melanoma growth in vivo, nude mice were injected with RTL1-overexpressing or RTL1-knockdown melanoma cells. The tumour size was detected every 3 days, and the tumour weights were obtained at 4 weeks after injection. The results showed that RTL1 overexpression increased the tumour size and weight, whereas knockdown of RTL1 had the opposite effects ( Figure 5A-5D). Furthermore, in an immunohistochemistry assay, we observed that the expression levels of RTL1, c-MYC, CYCLIN D1 and β-CATENIN in tumour tissues were increased after overexpression of RTL1 and decreased with RTL1 knockdown ( Figure 5E), in agreement with previous findings in A375 cells. Therefore, β-Catenin and Wnt/β-Catenin signalling are the main downstream targets of RTL1 both in vitro and in vivo.

DISCUSSION
Because melanoma is a highly aggressive skin cancer, the most effective period for curing melanoma is in early phases. Detection and control of melanoma in advance can greatly increase patient survival rate [31]. Intervening in melanoma proliferation is critical for the early treatment of melanoma. RTL1 is the key member of the Dlk1-Dio3 imprinted region, and the loss of imprinting in this region and network disorder of imprint regulation are closely related to tumour formation [32]. Previous studies have shown that RTL1 is involved in several cancers, including lung cancer [12], squamous cell carcinoma of head and neck [13], and hepatocellular carcinoma [14][15], and the methylation level of RTL1 in melanoma patients is significantly lower than people without cancer [33]. In this study, we provided evidence that RTL1 is highly expressed in human melanoma tissues and that the positive rate in early and horizontal growth melanoma is much higher than in vertical growth and metastasis states. Moreover, RTL1 was found to promote  the cell cycle transition from the G1 to the S phase and the proliferation of melanoma cells. These results suggested that RTL1 may be an important regulatory molecule in promoting early melanoma formation and development, and a potential predictor of melanoma. β-Catenin deposits into the nucleus, binds to TCF/ LEF-type transcription factors, and consequently activates downstream genes, such as Cyclin D1 and c-MYC [34]. In contrast, β-Catenin can be phosphorylated by casein kinase 1 and glycogen synthase kinase 3 in cytoplasm and subsequently degraded by the proteasome [35]. Abnormal activation of the Wnt/β-Catenin pathway has been reported as one of the predisposing factors in melanoma [19][20][21][22]. In the present study, we found that RTL1 overexpression activated the Wnt pathway, whereas knockdown of RTL1 inhibited the Wnt pathway. The inhibition of the nuclear accumulation of β-Catenin reversed the effects of RTL1 on melanoma cell proliferation, thus indicating that RTL1 promotes melanoma cell proliferation by modulating the Wnt/β-Catenin signalling pathway. Mechanistically, we found that RTL1 did not directly affect the expression of β-Catenin; instead, it increased the protein level of β-Catenin in melanoma cells by regulating the expression of DOCK4 and MACF1, both of which enhance the   release of β-Catenin from the destruction complex and increase the stability of β-Catenin [36].
Immuno-suppression and resistance are the main factors that negatively influence targeted therapies for melanoma [29,37]. Vemurafenib and Dabrafenib are two FDA-approved specific BRAF inhibitors and that are used to treat metastatic melanoma patients with BRAF mutations [38][39][40]. Although Vemurafenib rapidly improves the symptoms of melanoma patients, most patients acquire symptoms of drug resistance after 6-7 months [41][42][43]. Similar drug resistance has also been found in patients treated with an ERK inhibitor [44] or MEK inhibitor [45]. The occurrence of drug resistance in melanoma treatment has been found to be associated with multiple mechanisms, including secondary mutations, target gene mutations, changes in drug metabolism, and the activation of compensatory pathways [41]. Strikingly, Wnt/β-Catenin signalling appears to play a key role in immune resistance in melanoma. For instance, in melanoma with β-Catenin overexpression, T cells are prevented from entering the tumour tissue, and the infiltrated CD103 + dendritic cells are dramatically decreased [46]. Up-regulation of β-Catenin-LEF1 and concomitant down-regulation of YAP1 have been found to sensitize MAPKi-resistant melanoma [47]. In this study, we found that RTL1 activated the Wnt/β-Catenin pathway, thus suggesting that targeting RTL1 may provide a novel therapeutic target to melanoma patients or those with abnormally activated Wnt/β-Catenin signalling and related drug resistance.
In conclusion, we found that RTL1 is highly expressed in early rather than later stages of human melanoma tissues and that it critically regulates the cell cycle regulation and melanoma cell proliferation. Additionally, β-Catenin and Wnt/β-Catenin signalling are the main downstream targets of RTL1 both in vitro and in vivo. These findings indicate that RTL1 is involved in melanoma development and may serve as a potential new molecular target for melanoma diagnosis and therapeutics.

Cells and patients
Two melanoma cell lines A375 (a human malignant melanoma cells) and A875 (a melanoma cell line), a lung cancer cell line (A549) and a liver cancer cell line (HepG2) were used in the study. Also a keratinocytes cell line HaCat had been used. All the cell lines were purchased from the Cell Bank at the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences and cultured in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO, USA) supplemented with 10% foetal calf serum, 100 U/ml streptomycin and 100 U/ml penicillin. A total of 32 human patients with melanoma, 4 patients with nevus, 4 patients with other skin cancer (two basal cell carcinoma and two squamous cell carcinoma), and 4 controls with normal skin were collected from Shanghai Skin Disease Hospital in China with written informed consent. This work has been approved by the ethical committees of our institution. The age of patients with melanoma are from 11 to 84, and the average age is 53.03±16.82. There was 16 female patients and 16 male patients. In 32 patients, there were 4 patients with early or superficial melanoma, 4 patients with horizontal growth melanoma, 12 patients with vertical growth melanoma and 12 with lymph nodes metastasis.

Virus generation and infection
To generate lentiviruses, the plasmids carrying the specific sequences were transfected into HEK293T cells by the XtremeGENE HP DNA transfection reagent (Roche). After transfection for 48 h, the viruses were harvested. The cells were plated at a density of 1×10 5 cells/ well of 6-well plate and were infected with the viruses.

Cell proliferation and cell cycle analysis
Cell proliferation was evaluated on the basis of using cell counts and a Cell Counting Kit-8 (CCK8, DOJINDO, Japan), as measured at 0 h or 6 h, 24 h, 48 h, and 72 h following plating. Data were analyzed according to the manufacturer's instructions.
The cell cycle was analyzed by FACS. The cells were harvested by trypsinization after cultured for 12 h, then fixed in 75% alcohol for 30 min. After washed 3 times by PBS, the cells were resuspended in the PBS, stained with PI for 15 min at room temperature in the dark, and then kept on ice until analyzed.

Reverse transcription and quantitative real-time PCR
Total RNA was isolated from cells with TRIzol (Invitrogen, CA), and 500 ng of RNA was used to obtain cDNAs with the PrimeScript™ RT reagent Kit (TaKaRa, Japan). The cDNAs were used for real-time PCR analysis. The expression level of each gene was normalized to GAPDH and was further compared to the expression in the control group. The primers are shown in Supplementary

Tissue microarray and evaluation of immunostaining
Melanoma cancer tissue microarrays were purchased from the SuperBiotek Company in Shanghai, China. Institutional Review Board permission for the use of samples was obtained. The evaluation of expression was simultaneously made by two blinded independent observers, and a consensus score was recorded, according to the staining intensity and the percentage of positive cells.

Tumour xenografts in nude mice
The animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji University and complied with all regulatory guidelines. Four-week-old nude mice were purchased from the National Resource Center for Rodent Laboratory Animals of China. Five mice were subcutaneously injected with 1×10 6 cells of RTL1-overexpressing (RTL1-OE), RTL1knockdown (shRTL1), or control (FUGW, scramble) cells. The tumours were monitored, and the volumes were calculated at 3-day intervals after appearance.

Luciferase reporter gene assay
TOP flash and FOP flash constructs were used to evaluate β-catenin-dependent signaling events that drive the expression of TCF. TOP flash is a TCF reporter plasmid containing two sets of three copies of wildtype TCF binding sites driven by the thymidine kinase minimal prompter and upstream of a luciferase reporter gene. FOP flash contains mutated TCF binding sites driven by the same thymidine kinase promoter and also upstream of the same luciferase open reading frame as TOP flash. FOP flash was used as a negative control for TOP flash activity.
Cells were cultured in DMEM with 10% FBS and plated in 24-well plates at a density of 5×10 4 cells per well. For each well, cells were transfected with either 0.5 μg of TOP flash or 0.5 μg of FOP flash reporter plasmid together with 0.1 μg of TK-Renilla luminescent reporter plasmid using Lipofectamine LTX and PLUS reagents (Invitrogen, CA). The assays were performed by the Dual-Luciferase Reporter Assay System (Promega, WI), and data were adjusted on the basis of the Renilla activity in the same sample. Each corrected TOP flash luciferase value was compared with the corresponding corrected FOP flash value. Three independent treatments were performed.

Statistical analysis
The error bars represent the standard errors of the means (SEM) of three independent experiments. Data were tested for normalization and standard deviations to determine the appropriate statistical test (parametric versus non-parametric). Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001, or # p< 0.05, ## p < 0.01, and ### p < 0.001.

Author contributions
FGB performed the experiments and wrote the paper. YD analyzed some of the experimental results and provided technical assistance. ZSC, XJJ, GXD, QJ, WYK, and WWJ contributed to the preparation of the manuscript. WGY, FGH, and KJH conceived and designed the study and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

CONFLICTS OF INTEREST
No potential competing interest. www.impactjournals.com/oncotarget