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

Abstract

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.

Keywords: GSEA; Melanoma; Survival analysis; TCGA; UTRN.

Fig. 1

Decreased UTRN expression in melanoma. a Analysis of UTRN expression in nevus tissues (n = 18) and melanoma (n = 45) in GEO database (GSE3189) (P < 0.001). b Analysis of UTRN expression in normal skin (n = 812) and melanoma (n = 470) from UCSC XENA platform (P < 0.001). c, d Melanoma tissues (n = 20) presented lower expression of UTRN while benign nevus (n = 15) showed higher expression by IHC staining. e–g UTRN expression levels measured in 8 paired clinical samples by qRT-PCR (e) and Western Blot (f, g). IHC stain, AEC, scale bar represents 200 μm. Bars represent mean ± SD; n.s not significant, IHC immunohistochemistry. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

Fig. 2

Association with UTRN expression and patient clinical characteristics. The expression of UTRN was correlated to T stage (a), Clark level (b) and Ulceration (c) in melanoma patients in TCGA cohort. d, e Progression-free survival (d) and overall survival (e) curves for the patient groups with low and high UTRN mRNA levels were estimated by Kaplan–Meier survival analysis. TCGA The Cancer Genome Atlas. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

Fig. 3

UTRN inhibited proliferation via p38 and JNK1/c-Jun pathways in A375 melanoma cell. a–c qRT-PCR (a) and WB (b, c) results showed UTRN expression after stable transduction with lentiviral vectors. d CCK-8 assay of A375 cell line including EXP, EXP-NC and Blank control groups. e, f EdU incorporation assay in A375‑EXP‑UTRN transfected cells and negative controls. Magnification, × 200. g, h WB results showed that p38, JNK and c-Jun were down-regulated in EXP group. i After treatment with 8-pCPT-2′-O-Me-cAMP (100 μM, 30 min), the levels of p38, JNK and c-Jun were enhanced. j The proliferative ability was increased after 8-pCPT-2′-O-Me-cAMP treatment. 8-CPT: 8-pCPT-2′-O-Me-cAMP. Data was analyzed by the Student’s t-test. Bars represent mean ± SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001