Targeting PSMD14 inhibits melanoma growth through SMAD3 stabilization

Abstract

Although melanoma therapy is improved by novel molecular targeted reagents, including vemurafenib, aberrant proliferation and early metastasis remain obstacles for melanoma; therefore, novel target molecules for melanoma need to be identified. In this study, we focused on deubiquitinating enzymes, which regulate protein stability through ubiquitin-proteasome systems, and identified 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) as a molecule related to melanoma growth using siRNA library screening. Similar to a previous report, PSMD14 knockdown strongly induced p21 expression and inhibited RB phosphorylation in melanoma. After in silico analysis, TGF-β signaling was identified as a negatively correlated gene set with PSMD14 expression. Although TGF-β signaling is also related to the invasive phenotype of melanoma, PSMD14 knockdown suppressed melanoma migration and reduced SLUG expression, suggesting that targeting PSMD14 suppresses both growth and migration. Furthermore, SMAD3 expression increased in nucleus and SMAD3 degradation was delayed after PSMD14 knockdown. Thus, our present study suggests that targeting PSMD14 can inhibit melanoma growth and migration through either SMAD3 accumulation or SLUG reduction, respectively.

Figure 1

PSMD14 is related to melanoma growth. (A) UACC257 and M14 cells were transfected with a siRNA library against deubiquitinating enzymes for 96 h and subjected to the cell growth assay using WST-1. (B) UACC257 and M14 cells were transfected with siRNA against negative control (siCNTL), PSMD14 (siPSMD14), UBL5 (siUBL5), or BAP1 (siBAP1) for 96 h, and subjected to the cell growth assay using WST-1. (C) Human melanoma cell lines were transfected with each indicated siRNA for 96 h and subjected to the cell growth assay using WST-1. Data are shown as the mean ± SD of at least three independent experiments. *P < 0.01 vs siCNTL-transfected cells by one-way ANOVA followed by the Bonferroni post-hoc test. (D,E) UACC257 and M14 cells were transfected with each indicated siRNA for 96 h, and subjected to Western blotting (D) and real-time RT-PCR (E). *P < 0.01 vs siCNTL-transfected cells by one-way ANOVA followed by the Bonferroni post hoc test.

Figure 2

PSMD14 regulates SMAD3 stability in melanoma. (A) Top 1% of genes negatively correlated with PSMD14 were extracted from melanoma data in the Cancer Cell Line Encyclopedia. The extracted top 1% genes were analyzed by the gene set enrichment analysis using the Molecular Signatures Database. KEGG pathways significantly enriched are shown. (B) UACC257 and M14 cells were transfected with PSMD14 siRNA for 96 h. The whole cell lysates were subjected to Western blotting. (C) Nuclear or cytoplasmic protein was subjected to Western blotting. Other conditions were as in (B). (D) UACC257 cells were transfected with PSMD14 siRNA for 96 h. The transfected cells were treated with 50 μg/mL of cycloheximide for the indicated times and subjected to Western blotting. The band intensities were measured by ImageJ, normalized to that at 0 h for each cell lines, and shown below each panel.

Figure 3

Targeting PSMD14 inhibits melanoma migration and SLUG expression. (A) UACC257 cells were transfected with PSMD14 siRNA for 96 h and then subjected to the migration assay. *P < 0.01 vs siCNTL-transfected cells by one-way ANOVA followed by the Bonferroni post hoc test. (B) UACC257 cells were transfected with PSMD14 siRNA for 96 h. The whole cell lysates were subjected to the Western blotting.

Figure 4

PSMD14 regulates melanoma growth through the SMAD3-p21 axis. (A) UACC257 cells were transfected with the indicated siRNAs for 96 h. The chromatin immunoprecipitation assay was performed using the antibody against polymerase II phosphorylated serine 2 site (α-pol II S2) or SMAD3 (α-SMAD3). Immunoprecipitated DNA was quantified by real-time PCR using primers specific to the gene region of p21 or the SMAD-binding site on the p21 promoter. Results are normalized to the precipitated DNA using α-pol II S2 or α-SMAD3 in siCNTL-transfected cells. Data are presented as the mean ± SD of three independent experiments. *P < 0.01 vs. α-pol II S2-precipitated DNA or α-SMAD3 in siCNTL-transfected cells by two-way ANOVA followed by the Bonferroni post test. (B) UACC257 cells were transfected with the indicated siRNAs for 96 h. Relative mRNA expression was measured by quantitative real-time RT-PCR. (C) After 96 h, the transfected cells were subjected to the cell growth assay using WST-1. ^#P < 0.01 vs siCNTL/siPSMD14#19-transfected cells by one-way ANOVA followed by the Bonferroni post hoc test.