ERBB3 binding protein 1 promotes the progression of malignant melanoma through activation of the Wnt/ β-catenin signaling pathway

Abstract

Background: Malignant melanoma (MM) is highly metastatic and has the highest mortality rate in patients with skin cancer. The ERBB3 binding protein 1 (Ebp1) has been linked to the onset and progression of a number of malignancies. However, the role of Ebp1 in MM has not yet been reported.

Methods: Multiple databases were analyzed for comparing the expression of Ebp1 in normal skin and MM. Ebp1 expression was knocked down in A375 and B16 cells, and the impact of Ebp1 on the cell growth was tested by CCK-8, plate clone colony, and cell cycle assays. Scratch, transwell, and in vivo caudal vein lung metastasis tests were also used to confirm the effects of Ebp1 on melanoma cells migration, invasion, and metastasis. Furthermore, the possible molecular mechanism of Ebp1 was predicted by set enrichment analysis and verified by western blotting.

Results: Ebp1 expression was substantially higher in MM than it was in normal skin, and Ebp1 was linked to the clinical stage and lymph node metastases of patients with MM. Knockdown of Ebp1 inhibited cell proliferation, migration, and invasion. In vivo experiments further verified that the knockdown of Ebp1 had an obvious inhibitory effect on lung metastasis in nude mice. Knockdown of Ebp1 reduced vimentin, N-cadherin, slug, and snail expression while increasing E-cadherin expression. Furthermore, knockdown of Ebp1 reduced the expression of β-catenin, as well as its downstream targets CyclinD1 and p-GSK3β; however, a Wnt/β-catenin agonist could reverse this effect.

Conclusion: Ebp1 may promote the proliferation and metastasis of melanoma cells through activation of the Wnt/β-catenin pathway.

Keywords: EMT; Ebp1; Malignant melanoma; Wnt/β-catenin.

Fig. 1

Ebp1 is highly expressed in malignant melanoma and is closely related to prognosis. a 1809 normal skin tissue samples were analyzed in the GTEx database, the expression of Ebp1 was increased in 470 malignant melanoma tissue samples in the TCGA database. b The expression of Ebp1 was analyzed in tumor tissues of melanoma compared with normal skin tissue from the GEO database. c Kaplan–Meier analysis revealed high expression of Ebp1 correlates to poor overall survival in patients with malignant melanoma

Fig. 2

Ebp1 knockdown suppresses the proliferation of malignant melanoma. a Ebp1 p48 was expressed in the HacaT, A375 and B16 cells (^***p < 0.001, ^*p < 0.05, vs. HacaT). b, c Ebp1 was knocked down in A375 and B16 cells and detected by Western blotting (^****p < 0.0001, vs. shNC). d Cell proliferation was measured by CCK-8 assay (^**p < 0.01, ^*p < 0.05, vs. shNC). e Ebp1 knockdown suppressed the colony-forming capacity of A375 and B16 cells by colony-formation assay (^***p < 0.001, ^****p < 0.0001, vs. shNC). f Effects of Ebp1 knockdown on cell cycle profile in A375 and B16 cells using flow cytometry (^****p < 0.0001, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05, vs. shNC)

Fig. 3

Knockdown of Ebp1 inhibits proliferation and metastasis of melanoma cells in vitro and in vivo. a The migration capability of A375 and B16 cells were determined via wound-healing experiments (^***p < 0.001, vs. shNC). b Effect of Ebp1 on the invasion of melanoma cells by Transwell assay (^***p < 0.001, vs. shNC). c Ebp1 knockdown decreases the lung metastasis ability of B16 cells. d Morphological feature of the lung metastasis by H&E staining. e Surface pulmonary metastatic nodules were counted (^*p < 0.05, vs. shNC)

Fig. 4

Knockdown of Ebp1 inhibited EMT in melanoma cells. a Ebp1 was closely related to EMT by GESA analysis. b–d GEPIA website analysis indicated that the expression level of Ebp1 was positively correlated with CDH2 (N-cadherin), VIM (Vimentin) and SNAI2 (Slug). e The expression of EMT related proteins was detected by western blot (^***p < 0.001, ^**p < 0.01, ^*p < 0.05, vs. shNC). f, g Immunofluorescence staining assay was used to detect the expression of E-cadherin and Vimentin, Scale bar: 50 µm

Fig. 5

Knockdown of Ebp1 inhibited the Wnt/β-catenin signaling pathway. a–c GEPIA website analysis indicated that the expression level of Ebp1 was positively correlated with CTNNB1 (β-catenin), CCND1 (CyclinD1) and GSK3B (GSK3β). d Western blotting showed that knockdown of Ebp1 down-regulated the expression of β-catenin, CyclinD1 and p-GSK3β. e CHIR99021 reversed the changes caused by Ebp1 knockdown in related proteins. (^****p < 0.0001, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05, vs. shNC ^####p < 0.0001, ^###p < 0.001, ^##p < 0.01, ^#p < 0.05, vs. shEbp1)