Introduction
The incidence of melanoma is growing globally, with approximately 280,000 new cases diagnosed every year [
1]. Although accounts for a small proportion of cutaneous tumors, melanoma is responsible for the greatest number of skin cancer-related deaths and causes nearly 55,500 deaths annually [
2], which is mainly related to the distant metastatic spread of melanoma [
3]. Now, the initiation and development of melanoma were identified to be caused by dysfunctions of oncogenic and tumor suppressor pathway, and different biomarkers have been identified to be highly significant and relevant in the context of melanoma, including BRAF, NRAS, and C-KIT [
4‐
6]. Of these, 50% of melanomas harbor mutations in BRAF, mainly at codon 600, which results in the continuous activation of the MAPK pathway [
7]. Although targeted drugs, such as vemurafenib and dabrafenib, have improved the survival of melanoma patients, the prognosis remains dismal [
2]. Thus, it is critical to discover new biomarkers that drive the initiation and progression of melanoma, which may help to develop new targets for diagnosis and treatment.
Ubiquitination plays an essential role in protein posttranslational modification and is strongly linked to different biological and pathological processes in eukaryotes [
8]. Ubiquitin-protein enzymes (E3s) are of particular concern in this process, as they not only transfer activated ubiquitin from ubiquitin-conjugating enzymes to protein substrates but also confer substrate specificity [
9]. The RING finger protein family, a complex set of E3s that contain an RNF domain, was recently demonstrated to play crucial roles in tumorigenesis and tumor progression [
10]. For example, RNF13 is an ER/Golgi membrane-associated E3, and overexpressed RNF13 increases the invasive potential and gelatinolytic activity of pancreatic cancer by increasing matrix metalloproteinase-9 (MMP9) activity [
11]. Another example is RNF183, which contributes to the progression from inflammation to malignancy by activating the NF-κB-IL-8 axis in colorectal cancer [
12]. Identifying more cancer-related RNF family members will help us to better understand the mechanisms of tumor progression and develop new therapeutic strategies.
In the current study, we reanalyzed the available gene expression profiles from the GEO database and revealed that RNF128 was consistently downregulated in the selected datasets. Here, we also demonstrated that low level of RNF128 was closely related to Breslow depth, Clark level, distant metastasis, and TNM stage of melanoma. Moreover, RNF128 interference promoted cellular EMT and the acquisition of stemness by activating the Wnt pathway via ubiquitinating and degrading the CD44 and CTTN proteins, resulting in the transcription of CD44 and c-Myc, which indicated that RNF128 participated in a positive feedback of the Wnt signaling-CD44 loop. Thus, our study indicates that low level of RNF128 is a promoter of melanoma, and a deeper understanding of RNF128 may contribute to the diagnostic, prognostic, and therapeutic strategies.
Materials and methods
Data availability
To identify relevant RNF family members that are critical in the pathogenesis of melanoma, we performed data mining on the GEO database (
https://www.ncbi.nlm.nih.gov/gds/). GSE3189 was based on the GPL96 platform (HG-U133A, Affymetrix Human Genome U133A Array), and including 45 melanoma and 7 normal tissues, and GSE7553 was based on the GPL570 platform (HG-U133_ plus 2, Affymetrix Human Genome U133A Plus 2.0 Array), and including 14 melanoma and 4 normal tissues. GEO2R (
https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to calculate the adjusted
p values and logFC values among different groups. GSEA was performed using GSEA 2.2.1 (
http://www.broadinstitute.org/gsea).
Patients and follow-up
A total of 138 paraffin-embedded melanoma and matched peritumoral tissues and an additional 58 melanoma tissues were collected to construct the tissue microarray (TMA). Thirty pairs of frozen melanoma and matched nontumor tissues were randomly selected and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. All patients underwent curative resection verified by pathological examination at Zhongshan Hospital of Fudan University (Shanghai, China). Clinicopathological information was collected from 1 January 2008 to 31 December 2017. The Ethics Committee of the Zhongshan Hospital Biomedical Research Department provided ethical approval, and informed consent for collecting and preserving samples and details was obtained from each patient.
Cell culture and transfection
The human melanoma cell lines A2058, A375, A875, MV3, M14, and Sk-mel-28 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in DMEM or RPMI-1640 medium (HyClone, USA) with 10% fetal bovine serum (Invitrogen, USA), penicillin (100 IU/ml), and streptomycin sulfate (100 μg/ml) at 37 °C in a thermostatic incubator containing 5% CO2. pLVX-shRNA-eGFP-PGK-Puro and CMV-H_RNF128-eGFP-3flag-PGK-Puro lentiviral vectors were purchased from Genomeditech (Shanghai, China). The pLVX-shRNA-eGFP-PGK-Puro lentiviral vectors were transfected into M14 cells, and the pGMLV-SC5-Puromycin vectors were used as negative controls. The CMV-H_RNF128-eGFP-3flag-PGK-Puro lentiviral vectors were transfected into A2058 cells. siRNAs against Snail, CD44, CTTN, and β-catenin were designed and synthesized by Genomeditech (Shanghai, China). The target sequences were as follows: siSnail, GCGTGGGTTTTTGTATCCA; siCD44, CTGAAATTAGGGCCCAATT; siCTTN, CCTTAAGGAGAAGGAACTT; and siβ-catenin, TGGTTGCCTTGCTCAACAA. The siRNA was transfected using Lipofectamine™ 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s protocols. The efficiency of silencing was confirmed by western blot and qRT-PCR after 72 h of transfection.
TMA construction and IHC staining
The construction of TMA and immunohistochemistry (IHC) staining were performed as described previously [
13,
14]. Briefly, the slide was deparaffinized, rehydrated, subjected to antigen retrieval, and incubated in 0.3% H
2O
2. Subsequently, the sections were incubated with the primary antibody (listed in Additional file
1: Table S2) at 4 °C overnight and then stained with horseradish peroxidase-labeled IgG (Gene Tech, China). Then, the section was stained with diaminobenzidine, counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and cover-slipped. The density of positive staining was measured as previously described [
13]. Briefly, images of 4 representative fields were captured under high-power magnification (× 200), and identical settings were used for all of the images. The integrated absorbance and area of the images were counted by Image-Pro Plus v6.0 software (Media Cybernetics, Inc., Bethesda, MD, USA), and uniform settings were applied for all slides. The average density was calculated as the product of the integrated absorbance/total area, and the sections were classified as either high or low expression.
qRT-PCR and western blot analysis
Total RNA was extracted from both the tissues and cultured cells using TRIzol reagent (Invitrogen, USA) and reverse-transcribed to cDNA with a PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacturer’s instructions. The cycling conditions were 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Each reaction was performed in triplicate. The primer sequences for qRT-PCR are shown in Additional file
2: Table S1. Western blot was performed as described in a previous study [
15], and all the primary antibodies are listed in Additional file
1: Table S2.
Matrigel invasion and wound-healing migration
For invasion assays, cells were incubated using 24-well transwell plates (8-μm pore size, Corning, NY, USA). One million cells suspended in serum-free medium were plated in the upper chambers with Matrigel (BD Biosciences, USA), and 0.6 ml of DMEM or RPMI-1640 medium with 10% FBS was added to the lower chamber. After incubation for a suitable amount of time, the cells were fixed in 4% paraformaldehyde, stained by crystal violet, and counted under a microscope. For wound-healing migration assays, the cell monolayers were mechanically disrupted using a sterile 200-μl pipette tip to generate a linear wound. The average distance migrated by the cells was measured using a microscope calibrated with an ocular micrometer at a suitable time.
The colony formation assay was performed as described in a previous study [
16]. Briefly, cells were seeded in a 6-cm culture dish (1000 cells), and the culture medium was refreshed every 3 days for 2 weeks. After that, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.4% crystal violet for 15 min. The number of colonies containing > 10 cells was counted manually and averaged over duplicate wells. For sphere formation assay, cells were plated in ultralow attachment 6-well plate (Corning Inc., USA) at the density of 1000 cells per well in a 2 ml of serum-free DMEM/F12 basal medium supplemented with
l-glutamine (2 mM), 20 ng/ml human epidermal growth factor, 20 ng/ml human fibroblast growth factor-2, and B-27 supplement (1:50) at 37 °C for 2 weeks. After that, the diameters of each cell sphere were measured, and the numbers of the sphere with a diameter > 100 μm were counted as primary spheres. Cell proliferation was detected by the Cell Counting Kit-8 (CCK-8, Yeasen, Shanghai, China) and performed as described in a previous study [
17]. Briefly, cells were inoculated into 96-well plates (1000 cells per well). Then, 10 μl of CCK-8 reagent (Yeasen, Shanghai, China) was added to the wells after the first, second, third, and fourth days. The plates were incubated for 2 h, and the absorbance was determined at 490 nm.
Flow cytometric and immunofluorescence assays
Flow cytometric analysis was performed as in a previous study [
18] and was used to determine the percentage of positively stained cells. A total of 10
5 cells were collected in the tube and stained with Annexin V-APC/7-ADD (Yeasen, Shanghai, China) or PE-CD133 (BioLegend, CA, USA). Positively stained cells were quantified by flow cytometry (Becton Dickinson) and analyzed by FlowJo-V10 software. Immunofluorescence was used to detect the location and expression of target proteins, as described previously [
19]. Briefly, after being fixed with 4% paraformaldehyde, incubated in 0.3% Triton X-100 and blocked with 5% FBS, cells were incubated with primary antibodies at 4 °C overnight, followed by incubation with the appropriate secondary antibody (Yeasen, Shanghai, China). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Yeasen, Shanghai, China). The intensity of fluorescence was detected by confocal laser scanning microscopy (LSM510; Zeiss, Germany).
Xenograft experiments in nude mice were approved by the Animal Experimentation Ethics Committee of Zhongshan Hospital, Fudan University. Male BALB/c nude mice aged 4–6 weeks were maintained according to the stated guidelines of the 3 Rs (replacement, reduction, and refinement). All mice were randomized, and the investigators were blinded to the group assignment. We resuspended 106 cells (per mouse) in 100 μl of PBS and injected them into the lateral tail vein. The mice were sacrificed after 30 days; the lungs were resected, embedded in paraffin, and stained with hematoxylin and eosin (H&E); and lung metastases were counted.
IP assay and MS
Cells were harvested in lysis buffer supplemented with a protease inhibitor. After removing the insoluble material by 12,000×g centrifugation, the protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Precleared lysates with equivalent amounts of protein were incubated with a primary antibody overnight at 4 °C. Then, protein A- and G-Sepharose beads (Pierce Biotechnology, Rockford, IL, USA) were added to the immunoprecipitation (IP) mixture for 2 h. The precipitates were washed with lysis buffer three times, resuspended in SDS-PAGE sample buffer, boiled, and loaded onto 8% gradient gels. Subsequent western blots were probed with the target antibody and detected by enhanced chemiluminescence (Millipore, Darmstadt, Germany).
Mass spectrometry (MS) assay was performed to detect the potential interacting proteins, as described previously [
20]. Briefly, the immunoprecipitates described above were resolved on 10% gradient gels, and the protein bands of interest were visualized by Coomassie blue staining and used for MS analysis. MS was operated in reflection mode with an m/z range of 400 to 2000 and a 19 kV accelerating voltage. All MS data were identified using SEQUEST (v.28, Thermo Electron) against the Human International Protein Index database.
Ubiquitination assay and CHX chase assay
M14-shNC and M14-shRNF128 cells were transiently transfected with hemagglutinin (HA)-tagged ubiquitin vectors. Forty-eight hours later, the cells were washed with PBS and incubated with 10 μM MG132 (Selleck, Texas, USA) for 8 h. Then, the cells were lysed and subjected to immunoprecipitation with anti-CD44 and anti-CTTN antibodies. Ubiquitination of substrates was analyzed by SDS-PAGE after blotting with an anti-HA antibody. The half-life of CD44 and CTTN was determined by a CHX chase assay. M14-shNC and M14-shRNF128 cells were treated with cycloheximide (CHX) (100 mg/mL) for the indicated times, and western blot was performed to detect the expression of CD44 and CTTN.
TOP/FOP luciferase reporter assay
The TOPFlash/FOPFlash luciferase reporter assay was performed using the Luciferase Assay System (Promega) according to the manufacturer’s instructions. Briefly, cells were co-transfected with the Wnt/β-catenin signal pathway reporter TOPFlash/FOPFlash. After 24 h of plasmids transfection, cells were lysed and luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega). The luciferase activities of Firefly and Renilla were determined using a luminometer.
Statistical analysis
All in vitro experiments were repeated at least three times. The data were analyzed using IBM SPSS Statistics 20 (
IBM Corp., USA), and the values are presented as the mean ± standard deviation (SD). Student’s
t test or Tukey’s multiple comparisons test was used for comparisons between two groups, and one-way ANOVA was used for multiple group comparisons. Correlations between the two groups were detected by analysis of Pearson’s correlation coefficient. OS and recurrence rates were analyzed using the Kaplan-Meier method and the log-rank test. Independent prognostic factors were analyzed by Cox’s proportional hazards regression model. All statistical tests were two-sided, and differences were considered statistically significant at
p < 0.05.
Discussion
The prognosis of melanoma patients remains unsatisfactory [
2]. In the current study, we found that RNF128 expression was significantly downregulated in melanoma compared with that in matched peritumorous tissues, and the downregulation of RNF128 is strongly correlated with poor prognosis in melanoma patients. Moreover, we further demonstrated that low levels of RNF128 activated canonical Wnt signaling and participated in a positive feedback for the CD44-Wnt loop; thus, RNF128 functions as a tumor suppressor gene in melanoma.
RNF128 is a type I transmembrane ubiquitin-protein enzyme that localizes to the endocytic pathway [
24], which has previously been reported to associate with innate and adaptive immune responses. For example, Song et al. demonstrated that RNF128 promotes IRF3 activation, IFN-β production, and innate antiviral immune responses to RNA and DNA viruses [
25], and Roza et al. found that RNF128 regulation is critical for naïve T cell tolerance and regulatory T cell function, as evidenced by the greatly increased susceptibility to autoimmune diseases in Rnf128
−/
− mice [
26]. Recently, RNF128 was confirmed to play important roles in tumorigenesis and progression. Chen et al. found that RNF128, as a tumor promoter, physically interacts with and degrades p53 under stress conditions [
27]. Conversely, Lee et al. found that downregulation of RNF128 was associated with the reduced survival in patients with urothelial carcinoma [
28]. Here, we found that RNF128 expression is downregulated in melanoma compared with that in adjacent peritumoral tissues. Low levels of RNF128 were shown to induce melanoma cell EMT and promote lung metastasis through the Wnt/β-catenin pathway via the ubiquitination of CD44 and CTTN. We provide compelling evidence for the role of RNF128 in tumorigenesis and progression. Many factors may lead to the low expression of RNF128, such as methylation, miRNA regulation, and so on; further study of the upstream regulatory mechanism of RNF128 will undoubtedly be helpful to the treatment of melanoma. What is more, a better understanding of the downstream signaling pathway of RNF128 will also contribute to the therapy of melanoma.
As an E3, RNF128 physically interacts with multiple target proteins for ubiquitination and proteasomal degradation. For example, RNF128 was reported to interact with TBK1 and catalyze the K63-linked polyubiquitination of TBK1, which led to IRF3 activation and IFN-β production [
25]. In our study, a combination of Co-IP and MS identified 10 proteins that might be substrates of RNF128. Among these substrates, multiple cytoskeletal regulatory proteins were found, which indicated that RNF128 plays a crucial role in cytoskeletal reorganization. These results were consistent with the previous results [
29]. However, some known partners of RNF128 in lymphocytes were not found in these tumor cell lines, possibly because RNF128 plays a different role in cancer cells. Here, we focused on CD44 and CTTN due to their high abundance in the interactome and their known oncogenic properties [
30,
31]. From the GEPIA analysis, the mRNA levels of these factors were slightly downregulated in tumor tissues compared with those in normal tissues (Additional file
9: Figure S7), which indicated that posttranslational modification plays crucial roles in CD44 and CTTN expression. Furthermore, when RNF128 was combined with CD44 or CTTN, RNF128-induced EMT was not evident, but when RNF128 was combined with both factors, RNF128 significantly promoted melanoma cell EMT, which indicated that these proteins play a synergistic role in activating downstream signaling pathways in melanoma cells.
EMT has been reported to be induced by multiple pathways, such as the Wnt, MAPK, and PI3K pathways [
32‐
34]. We found that CD44 and CTTN levels were positively correlated with the Wnt and MAPK pathways, as indicated by GSEA. By western blot, we found that the RNF128/CD44/CTTN complex could activate the Wnt and MAPK pathways. Using inhibitors of Wnt and ERK1/2, we found that inhibition of Wnt signaling powerfully repressed the EMT phenotype. It has been demonstrated that activation of the Wnt pathway can induce EMT in multiple tumors, including melanoma [
35‐
37]. Moreover, we showed that the expression of target molecules of Wnt signaling, such as CD44 and c-Myc, was regulated by RNF128 at both the mRNA and protein levels. Additionally, this upregulation of CD44 and CTTN activated the Wnt pathway and further upregulated the expression of CD44, which is a widely accepted marker in stem tumor cells [
38]. Interestingly, we found that a low level of RNF128 promoted stemness in melanoma cells, which was consistent with the results of the colony formation and sphere formation assays. Thus, downregulation of RNF128 activates Wnt signaling via CD44 and CTTN ubiquitination and is involved in a positive feedback of Wnt signaling-CD44 loop.
In conclusion, we provide a reliable molecule that can be used as a potential diagnostic biomarker, prognostic indicator, and even contribute to the treatment of melanoma.