UTRN inhibits melanoma growth by suppressing p38 and JNK/c-Jun signaling pathways

Background Utrophin (UTRN), as a tumor suppressor gene, is involved in various cancer progression. The function of UTRN in the melanoma process and the related molecular mechanisms are still unclear. Herein, we studied the function of UTRN in melanoma growth and the relevant molecular mechanisms. Methods Using the GEO database and UCSC Xena project, we compared the expression of UTRN in non-cancerous and melanoma tissues. Immunohistochemistry (IHC) staining, qRT-PCR and Western Blot (WB) were performed to evaluate UTRN expression in clinical samples. A total of 447 cases with UTRN expression data, patient characteristics and survival data were extracted from TCGA database and analyzed. After stable transduction and single cell cloning, the proliferation ability of A375 human melanoma cells was analyzed by Cell Counting Kit‑8 (CCK) and 5‑ethynyl‑2′‑deoxyuridine (EdU) incorporation assays. GSEA was performed to predict the mechanism by which UTRN regulated melanoma growth. Then WB analysis was used to assess the protein expression levels of pathway signaling in overexpression (EXP) melanoma cells. Epac activator 8-pCPT-2′-O-Me-cAMP was then used to evaluate the proliferation ability by activation of p38 and JNK/c-Jun signaling pathways. Results Data from GEO and UCSC Xena project indicated that UTRN expression was decreased in melanoma. Experiment on clinical samples further confirmed our finding. TCGA results showed that a reduced expression of UTRN in 447 melanoma samples was associated with advanced clinical characteristics (T stage, Clark level, ulceration), shorter survival time and poorer prognosis. In addition, up-regulated UTRN expression inhibited melanoma cell proliferation when compared to control group. MAPK signaling pathway was presented in both KEGG and BioCarta databases by using GSEA tool. WB results confirmed the down-regulated expression of p38, JNK1 and c-Jun in EXP group when compared to control group. Epac activator 8-pCPT-2′-O-Me-cAMP treatment could partially rescue proliferation of tumor cells. Conclusion We have demonstrated that reduced UTRN predicted poorer prognosis and UTRN inhibited melanoma growth via p38 and JNK1/c-Jun pathways. Therefore, UTRN could serve as a tumor suppressor and novel prognostic biomarker for melanoma patients.

on the clinical stage at diagnosis, 5-year survival rate ranges from 15 to 60% in patients with distant and local metastases [1][2][3]. Survival rate was associated with tumor characteristics such as Clark level, ulceration and lymph node involvement [4][5][6]. However, an effective biomarker which can further improve the accuracy of the prognostic indicators mentioned above is still needed. The aim of this study was to contribute to the search for a prognostic molecular marker in melanoma.
UTRN, which is located on the chromosomal band 6q24, encodes a component of cytoskeleton utrophin [7]. UTRN plays a role in the organization of connection between cytoskeleton and transmembrane proteins, and can compensate for dystrophin-deficient muscular dystrophy [8]. The expression level of UTRN depends on cell type and tumor. UTRN was found to be significantly decreased and associated with progression in lung cancer and colon cancer, which indicated that UTRN could be a potential tumor suppressor gene [7]. Recent studies also identified that UTRN was decreased in melanoma [7,9,10]. However, the link between UTRN and clinical outcome as well as the molecular functional role of UTRN in melanoma have not been made clear.
This work revealed the clinical significance of UTRN expression in melanoma and explored the underlying molecular mechanism. We first clarified that UTRN expression was decreased in melanoma by examining public database and clinical specimens. Then, we analyzed the correlation between UTRN and clinical characteristics as well as survival time in The Cancer Genome Atlas (TCGA). Finally, we found out that UTRN inhibited melanoma growth via p38 and JNK/c-Jun pathways and could act as a tumor suppressing gene.

Patients tissue specimens
Matched pairs of tumor and para-cancer tissues were collected from 8 melanoma patients of the Han Chinese group from 2017 to 2019 (Additional file 1: Table S1). All the patients were proved to have melanoma by histological examination.

Cell culture
A375 human melanoma cell line was obtained from American Type Culture Collection (ATCC). A375 was maintained in the DMEM (Gibco) supplemented with 10% FBS and cultured at 37 °C in a 5% CO 2 incubator.

Immunohistochemistry (IHC) staining
A human tissue microarray (TMA, Cat.# ME1004g, Alenabio) was constructed with formalin-fixed paraffinembedded melanoma tissues and nevus tissues. IHC staining was performed on the TMA by using the appropriate dilatation of primary antibodies (UTRN, 1:600, ab95443, Abcam) followed by incubation with a secondary antibody conjugated with HRP (mouse anti-rabbit, 1:5000, Abcam). TMA was then washed three times with PBS for incubation with AEC (ZSGB-BIO).

Histologic scoring and analysis
TMA was examined by the cross-product (H score) of the percentage of tumor cell staining at each of the 3 staining intensities. Intensity of immunopositivity was scored as follows: none, 0; weak, 1; moderate, 2; and strong, 3. For example, a particular tumor may have 50% cell staining at intensity = 1 and 50% of cell staining at intensity = 3, for a combined H score of 200 [(50 × 1) + (50 × 3) = 200], which yielded a range from 0 to 300. The final score was graded by H score as follows: Low, H score 0-100; Moderate, H score 101-200; and High, H score 201-300.

Stable transduction with lentiviral vectors and single cell cloning
The procedures of transduction with lentiviruses and single cell cloning have been described in our previous work [11]. Lentiviruses expressing overexpression (EXP) of UTRN were purchased from Cyagen Bioscience.

Cell proliferation assay
The cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer's specification. Briefly, 5 × 10 3 A375 cells per well were seeded in 96-well plate. To test the growth rate at 24 h, 48 h and 72 h, the number of viable cells in quadruplicate wells was determined using the CCK-8.

5-Ethynyl-2′-deoxyuridine (EdU)-incorporation assays
An EdU labeling kit (cat. no. C 10310-3; Guangzhou RiboBio Co., Ltd.) was used to investigate cell viability and proliferation in UTRN overexpression melanoma cells. 50 μM EdU labeling media was added in 96-well plate with stable transduction A375 cells. After fixed with 4% paraformaldehyde, the cells were immunostained with Apollo working solution. Then, the cell nuclei were stained with DAPI solution according to the manufacturer's instructions (cat. no. DA 0001; Beijing Leagene Biotechnology Co., Ltd.). The EdU-positive cells and DAPI cells in each field were counted and the proliferation rate was calculated from the formula: Proliferation rate = the green fluorescence-positive cells (proliferating cells)/the blue fluorescence cells (total cells) × 100%.

Microarray data and RNA-sequencing patient data
The gene expression profiles of melanoma were obtained from the Gene Expression Omnibus (GEO, available at http://www.ncbi.nlm.nih.gov/geo/) database. A previous microarray (GSE3189) data which compared the expression of UTRN between melanoma (n = 45) and nevus (n = 18) patients was downloaded from GEO database. Data was excluded with less than 10 samples. TCGA and GTEx RNA-Seq data were processed by the Toil pipeline and downloaded from the UCSC Xena project (https :// xenab rowse r.net/datap ages/). Wilcoxon rank sum test was performed to compare the expression of UTRN in normal samples obtained from GTEx and TCGA (n = 812) and melanoma samples from TCGA (n = 470).
Clinical information and the RNA-Seq data (470 cases) for melanoma were downloaded from TCGA database (https ://genom ecanc er.ucsc.edu). Patients with incomplete RNA-Seq data (2 cases) or cases with overall survival time less than 1 month (21 cases) were excluded from the analysis. Overall, 447 melanoma cases were enrolled in the study. RNA-Seq data were converted to TPM format and UTRN expression level was retained for further analysis.

Bioinformatics analysis
The correlation between clinical characteristics and UTRN expression in melanoma patients was analyzed by the Wilcoxon signed-rank test. The samples were then divided into a high and a low UTRN group based on the median value of UTRN. Logistic regression analysis was performed to determine the correlation between UTRN expression and clinical characteristics in the TCGA cohorts. Univariate and Multivariate Cox analysis were used to compare the influence of UTRN expression on survival time along with other clinical characteristics (TNM stage, clinical staging, Clark level, age, ulceration, recurrence, cancer status and race). Significant variables (P ≤ 0.05) found in univariate analysis were further tested in multivariate analysis.

Gene set enrichment analysis (GSEA)
Using the data obtained from TCGA, GSEA was performed to identify gene sets and pathways associated with UTRN. The expression profile data of 447 cases was sorted by UTRN gene expression value. The melanoma samples were divided into a low expression group and a high expression group according to the median value of UTRN expression. Gene set permutations were performed 1000 times for each analysis. The expression level of UTRN was used as a phenotype label. A normalized enrichment score (NES) is calculated for each gene set based on the size of the set. The differences were considered statistically significant if nominal P value ≤ 0.05 and false discovery rate (FDR) ≤ 25%.

Statistical analysis
Most of the statistical analyses were done by the bioinformatic tools mentioned above. Statistical analysis for experimental assay was performed with GraphPad Prism 7.0. Differences between the two groups were examined using Student's t-test. Variables with P value ≤ 0.05 were considered statistically significant (*P ≤ 0.05, **P ≤ 0.01, *** ≤ 0.001).

Decreased UTRN expression in melanoma
To evaluate UTRN expression in melanoma tissues, we first analyzed UTRN mRNA expression from GEO database (GSE3189) (Fig. 1a) and UCSC XENA platform (Fig. 1b). These data showed that UTRN mRNA level was significantly lower in melanoma compared with mRNA level in nevus tissues and normal skin. Next, we evaluated UTRN expression on TMA. The results showed that melanoma tissues presented lower expression of UTRN (n = 20) while benign nevus (n = 15) showed higher expression (Fig. 1c, d). Furthermore, qRT-PCR and WB were conducted to validate UTRN mRNA and protein levels in 8 paired samples of tumor and para-cancerous tissues ( Fig. 1e-g, Additional file 5). The results confirmed that UTRN mRNA and protein expression were both decreased in melanoma tissues compared to nontumorous tissues.

Patient characteristics
As shown in Table 1, data of 447 melanoma patients with clinical characteristics was downloaded from TCGA. Current prognostic model based on the classic tumor-node-metastasis (TNM) staging is the recognized prognosis standard for melanoma in clinical practice [12]. According to the T-staging classification, 7 patients (0.02%) were in stage Ti (0.02%), followed by 41 (11.48%)

Association between UTRN expression and patient clinical characteristics
A total of 447 samples from TCGA with UTRN expression and clinical characteristics were analyzed. As shown in Fig. 2a-c, UTRN expression level was associated with clinical characteristics including T stage (T1T2 vs. T3T4, P < 0.01), Clark level (I/II/III vs. IV/V, P < 0.001) and ulceration (No vs. Yes, P < 0.01) in melanoma patients. As shown in Table 2 Notably, ulcerated melanomas have been proved to be significantly associated with more metastasis and shorter survival time [16,17]. However, no significant differences were found in N stage (N0 vs. N1N2), M stage (M0 vs. M1), site (extremities vs. trunk), age (< 60 vs. ≥ 60), gender (female vs. male), height (< 170 vs. ≥ 170), cancer status (tumor free vs. with tumor), race (Asian vs. White), radiation therapy (No vs. Yes) and weight (< 80 vs. ≥ 80). These results suggested that UTRN expression was related to melanoma progression.

Survival outcomes and multivariate analysis
As shown in Fig. 2d, e, lower UTRN expression was found to be associated with poorer progression-free survival (PFS) (P = 0.018) and overall survival (OS) (P < 0.001) when compared with those with higher UTRN expression by Kaplan-Meier survival analysis.
The univariate analysis ( Taken together, these results indicated that UTRN level could serve as an independent prognostic factor for melanoma patients.

UTRN inhibited proliferation of melanoma cells
After stable transduction and single cell cloning, qRT-PCR and WB tests showed that mRNA and protein expression of UTRN were prominently altered in EXP group compared to control group (Fig. 3a-c). These successfully constructed recombinant lentiviral vectors lay down a strong foundation for further investigation into the possible role of UTRN in melanoma development. We next examined the effects of UTRN on cell proliferation. CCK-8 and EdU incorporation assays were used to study the effect of UTRN expression on melanoma cell proliferation. As shown in Fig. 3d-f, cell viability was significantly decreased in EXP group compared to control group.

UTRN down-regulated p38 and JNK/c-Jun molecular signaling in melanoma
To investigate the function of UTRN in melanoma, the effects of UTRN expression on various biological functional gene sets were analyzed by the GSEA approach. In the GSEA analysis of KEGG, BioCarta and Reactome enrichment (Additional file 2: Figure S1, Additional file 3:  Table S2), higher UTRN expression was involved in TGF-beta signaling pathway in KEGG enrichment analysis, Wnt signaling pathway in KEGG enrichment analysis, ErbB signaling pathway in KEGG enrichment analysis, MAPK signaling pathway in KEGG and BioCarta enrichment analysis, apoptosis pathway in KEGG enrichment analysis, Fas pathway in BioCarta enrichment analysis, PTEN pathway in Bio-Carta enrichment analysis, pathways in cancer in KEGG enrichment analysis, Toll-like receptor signaling pathway in KEGG, BioCarta and Reactome enrichment analysis, T cell receptor signaling pathway in KEGG enrichment analysis, B cell survival pathway in BioCarta enrichment analysis, ILS signaling pathway in Reactome enrichment analysis and IL2RB pathway in BioCarta enrichment analysis. Notably, it could be seen that MAPK signaling pathway was presented in both KEGG and BioCarta databases.
We subsequently identified hub genes in MAPK pathways and confirmed their differential expression. We focused on MAPK pathway which includes the signaling molecules p38, JNK and c-Jun. WB assay was performed and the results confirmed down-regulated expression of p38, JNK1 and c-Jun in UTRN-EXP group when compared to control group (Fig. 3g, h). Then, cells were treated for 30 min with the Epac-selective agonist (8-pCPT-2′-O-Me-cAMP, 100 μM) as a p38, JNK/c-Jun activator to rescue p38, JNK1 and c-Jun expressions [11].

Table 2 Logistic regression analysis for UTRN expression and clinical characteristics in TCGA patients
The univariate analysis investigated that UTRN expression was significantly correlated with T stage, Clark level and ulceration in melanoma patients. Categorical dependent variable, greater or less than the median expression level   The results indicated that upon addition of activator, p38, JNK1 and c-Jun were upregulated (Fig. 3i). To further verify whether UTRN mediated melanoma proliferative phenotype through p38, JNK/c-Jun pathways, we investigated cell proliferation activity when exposed to 8-pCPT-2′-O-Me-cAMP. This treatment could partially rescue cell proliferation by altering p38, JNK/c-Jun activity (Fig. 3j). These data confirmed our hypothesis that p38, JNK/c-Jun activity was responsible for UTRN-mediated growth in melanoma.

Discussion
UTRN, an autosomal homologue of dystrophin, is expressed in several tissues especially in mature skeletal muscle [18]. Recent studies have shown that UTRN plays an important role in various important cellular processes, such as the development of neuromuscular junctions, cell signaling, adhesion and apoptosis [7,18,19]. However, the link between UTRN and clinical characteristics as well as its biological function in melanoma has remained unclear.
Our study further confirmed that UTRN expression was decreased in melanoma. Additionally, bioinformatic analysis demonstrated that decreased expression of UTRN in melanoma was associated with advanced clinical characteristic (T stage, Clark level, ulceration), lower survival time and poorer prognosis. Functionally, up-regulated UTRN inhibited proliferation in A375 melanoma cell. To identify the underlying mechanism of UTRN in melanoma, GSEA using TCGA data and experimental assays were performed to elucidate that UTRN might regulate melanoma growth through p38 and JNK/c-Jun pathways.
Recent studies have demonstrated that UTRN levels were decreased in various cancers including lung cancer, colon cancer, stomach cancer, etc. [7]. Interestingly, some studies showed that UTRN expression was higher in the plasma of breast cancer patients and ovarian cancer patients, and UTRN membranous localization was significantly increased in pancreatic endocrine tumor [20,21]. Moreover, another research showed that UTRN expression did not change in peripheral T-cell lymphomas compared to that in the normal T-cells [22]. These results indicated that UTRN expression depended on the type of cancer and cells. Some studies also illustrated UTRN expression level in melanoma. Li et al. [7] reported that inactivation mutations of UTRN were found in melanoma cases (4/15). Another study showed that UTRN was a down-regulated gene by using microarray data from four melanoma-derived cell line pairs [9]. Similar results were reported in a study using microarray-based CGH [10]. Consistent with these results, we confirmed that melanoma tissues presented lower UTRN expression levels than non-tumorous tissues.
Ulceration in melanoma has been well-recognized as a prognostic factor for melanoma candidate selection [16,23]. Ulcerated melanoma is associated with a higher risk of metastasis, a lower OS and PFS in patients. The most recent version of the Staging System proposed by the American Joint Committee on Cancer (AJCC8) has concluded that ulceration is an independent adverse prognostic factor in melanoma [24]. In our study, UTRN expression was significantly correlated with ulceration. Previous studies reported that race and age could also be important factors which affected melanoma patients' prognosis [25]. Asian people are more likely to have a lower 5-year survival rate than White people, which is most likely due to delay diagnosis. Elderly people tend to have a more aggressive primary tumor, which present thicker depth and higher mitotic rates. Consistent with these results, our data confirmed that Asian people and elderly people had a poorer outcome when compared to White people and younger people, respectively.
The loss of heterozygosity (LOH) is a common occurrence in human tumors, which indicates the absence of a functional tumor suppressor gene in the lost region. Higher LOH rate was found at UTRN, which was proved to be significantly associated with poor prognostic factors including high TNM stages, high T stages, positive lymph node status, an unfavorable disease course, and the presence of > 10% solid growth pattern [26]. This finding strongly suggested that UTRN could function as a prognostically important tumor suppressor gene. Consistent with this function, a study showed that overexpression of UTRN resulted in inhibition of tumor cell growth [7]. However, another study showed that membranous UTRN was related to higher Ki-67 proliferative indices in pancreatic endocrine tumors [21]. In our study, up-regulated UTRN expression inhibited the proliferation of human melanoma cells. Taken together, maintaining UTRN activity was important for cell function.
To further investigate the functional mechanism of UTRN in melanoma, GSEA using TCGA data revealed that UTRN might be associated with MAPK signaling pathway. The findings of immunoblot assays demonstrated that upregulated UTRN inactivated p38 and JNK/c-Jun pathways, which were the two MAPK classical signal transduction pathways. 8-pCPT-2′-O-Me-cAMP, a specific activate regulator of Epac, has been proved to be upregulated p38 and JNK/c-Jun expressions successfully [11]. After exposed to 8-pCPT-2′-O-Me-cAMP, our results confirmed that p38, JNK/c-Jun activity was responsible for UTRN-mediated growth in melanoma. Recent research revealed that MAPK signaling pathway played crucial roles in the pathogenesis of melanoma and most melanomas exhibited constitutive activation of the MAPK pathway [27]. JNK activation was associated with cell proliferation and a shorter relapse-free period in malignant melanoma [28]. Conversely, inhibition of JNK/c-Jun signaling induced cell apoptosis [29]. In addition, activated p38 contributed to cell migration and in vivo growth of melanoma [30], while inhibition of p38 reduced melanoma cell proliferation [31]. Intriguingly, one study showed that SAPK/p38-MAPK signaling pathways were constantly activated in the hearts of utrophin-dystrophin knockout mice [32]. Similarly, our study also showed that upregulation of UTRN induced a reduction in the protein expression of p38, which indicated that p38 could act as a candidate protein for UTRN-modulated biological process.
This study had several limitations. The clinical information from the TCGA database was not comprehensive. Some information of melanoma patients, such as treatment information, tumor size and histological differentiation, was not available on the TCGA website. In future study, more clinical cases with follow-up and complete information will be collected to conduct a more comprehensive analysis of UTRN in melanoma.

Conclusions
In conclusion, our study revealed that UTRN was decreased in melanoma, and lower expression of UTRN was related to advanced clinical characteristic and unfavorable prognosis. Moreover, experimental assays identified that UTRN inhibited melanoma proliferation ability. In addition, p38 and JNK/c-Jun signaling pathways were the key biological mechanisms regulated by UTRN in melanoma. Collectively, this work discovered that UTRN could act as a tumor suppressor gene in melanoma and serve as a prognostic factor.