SIRT1 induces epithelial-mesenchymal transition by promoting autophagic degradation of E-cadherin in melanoma cells

Abstract

Melanoma is highly metastatic, and understanding of its molecular mechanism is urgently needed for the development of therapeutic targets and prognostic assessment for metastatic melanoma. SIRT1 is a nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacetylase, belonging to the mammalian sirtuin family. It has been reported that SIRT1 is associated with metastasis in various cancers. However, the molecular mechanism of SIRT1 in melanoma metastasis remains to be clarified. Here we report that SIRT1 induces the epithelial-mesenchymal transition (EMT) by accelerating E-cadherin degradation via autophagy and facilitates melanoma metastasis. Initially, we found that SIRT1 expression was frequently elevated in metastatic melanoma compared with primary melanoma. In addition, SIRT1 induced the EMT and promoted cell migration and invasion by decreasing E-cadherin expression. Further work demonstrated that SIRT1 accelerated the autophagic degradation of E-cadherin through deacetylation of Beclin 1. In addition, inhibition of autophagy recovered E-cadherin expression and suppressed cell migration and invasion by delaying the degradation of E-cadherin in SIRT1-overexpressing cells. Overall, our findings reveal a novel molecular mechanism for SIRT1 in melanoma metastasis, indicating that SIRT1 may serve as a viable therapeutic target for metastatic melanoma.

Fig. 1. Overexpression of SIRT1 in metastatic melanoma

a A analysis of SIRT1 mRNA expression in primary melanoma (n = 103) and metastatic melanoma (n = 370). The RNA data were downloaded from the TCGA database. b, c Analysis of SIRT1 mRNA levels in primary and metastatic melanoma tissues from the GSE46517 and GSE8401 public data sets. d The expression of SIRT1 in the primary and metastatic melanoma tissues was analyzed by IHC. e The relationship between SIRT1 expression and the melanoma tissue type was evaluated using Pearson’s χ² test

Fig. 2. SIRT1 promotes the EMT and increase metastatic potential in melanoma cells

a Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were harvested and protein expression was detected by western blot analysis. b, c Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were plated in the upper chamber of the transwell filters for 24 h. Cells migrating to the underside of the transwell insert were then counted. d A375-shSIRT1 and control cells were harvested, and protein expression was detected by western blot analysis. e, f A375-shSIRT1 and control cells were plated in the upper chamber of the transwell filters for 24 h. Cells migrating to the underside of the transwell insert were then counted. g Immunofluorescence staining of actin filaments (Phalloidin) in Sk-Mel-28 cells stably expressing either vector or Flag-SIRT1. Bar = 10 μm

Fig. 3. SIRT1 promotes the lysosomal degradation of E-cadherin

a–c A375 cells were transfected with the indicated concentrations of Flag-SIRT1 for 24 h. Protein expression were analyzed by western blotting and E-cadherin mRNA expression was analyzed by RT-PCR. d, e A375-shSIRT1 and control cells were treated with CHX (20 μM) for the indicated times, and the protein levels were detected by western blotting. f, g A375 cells were treated with MG132 (10 μM) for 2 h, and then also treated with CHX (20 μM) for the indicated times. The protein levels were detected by western blotting. h, i A375 cells were treated with CQ (100 μM) for 12 h, and then CHX (20 μM) was added for the indicated times. The protein level were detected by western blotting. The bars represent mean ± SE (n = 3). N.S nonsignificant; CHX cycloheximide; CQ chloroquine

Fig. 4. SIRT1 deacetylates Beclin 1 and stimulates autophagic degradation of E-cadherin

a The protein expression in Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were extracted and immunoprecipitated using an anti-acetylated lysine antibody. b The protein expression in A375-shSIRT1 and control cells were extracted and immunoprecipitated using an anti-acetylated lysine antibody. c Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were transfected with mCherry-EGFP-LC3 for 24 h. The cells were then imaged by confocal microscopy. Scale bars, 5 μm. d Quantitation of the red and yellow puncta in c. The bars represent mean ± SE of 50 cells; three independent experiments, **P < 0.01 (Student’s t test). e Sk-Mel-28 cells expressing either vector or Flag-SIRT1 were treated with or without CQ (100 mM) for 12 h, and protein expression was measured by western blotting. f Sk-Mel-28 cells expressing either vector or Flag-SIRT1 were transfected with HA-tagged Beclin 1 (WT, 2KR) for 24 h, and protein expression was then measured by western blotting. AceK, acetylated lysine

Fig. 5. SIRT1 promotes cell migration and invasion by autophagic degradation of E-cadherin

a, b Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were plated in the upper chamber of transwell filters for 24 h and then treated with PBS or CQ (100 mM, 12 h). Cells migrating to the underside of the transwell insert were subsequently counted. c Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were treated with PBS or CQ (100 mM, 12 h), and protein expression was measured by western blotting. d, e Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were transfected with HA-tagged Beclin 1 (WT, 2KR) for 24 h. Cells were plated in the upper chamber of transwell filters for 24 h, and then cells migrating to the underside of the transwell insert were counted. f Sk-Mel-28 cells stably expressing vector or Flag-SIRT1 were transfected with HA-tagged Beclin 1 (WT, 2KR) for 24 h, and protein expression was then measured by western blotting

Fig. 6

Schematic diagram of the mechanism of SIRT1-mediated autophagic degradation of E-cadherin which stimulates the EMT in melanoma cells