Melatonin inhibits ESCC tumor growth by mitigating the HDAC7/β-catenin/c-Myc positive feedback loop and suppressing the USP10-maintained HDAC7 protein stability

Abstract

Background: Melatonin, a natural hormone secreted by the pineal gland, has been reported to exhibit antitumor properties through diverse mechanisms of action. However, the oncostatic function of melatonin on esophageal squamous cell carcinoma (ESCC) remains elusive. This study was conducted to investigate the potential effect and underlying molecular mechanism of melatonin as single anticancer agent against ESCC cells.

Methods: ESCC cell lines treated with or without melatonin were used in this study. In vitro colony formation and EdU incorporation assays, and nude mice tumor xenograft model were used to confirm the proliferative capacities of ESCC cells. RNA-seq, qPCR, Western blotting, recombinant lentivirus-mediated target gene overexpression or knockdown, plasmids transfection and co-IP were applied to investigate the underlying molecular mechanism by which melatonin inhibited ESCC cell growth. IHC staining on ESCC tissue microarray and further survival analyses were performed to explore the relationship between target genes' expression and prognosis of ESCC.

Results: Melatonin treatment dose-dependently inhibited the proliferative ability and the expression of histone deacetylase 7 (HDAC7), c-Myc and ubiquitin-specific peptidase 10 (USP10) in ESCC cells (P < 0.05). The expressions of HDAC7, c-Myc and USP10 in tumors were detected significantly higher than the paired normal tissues from 148 ESCC patients (P < 0.001). Then, the Kaplan-Meier survival analyses suggested that ESCC patients with high HDAC7, c-Myc or USP10 levels predicted worse overall survival (Log-rank P < 0.001). Co-IP and Western blotting analyses further revealed that HDAC7 physically deacetylated and activated β-catenin thus promoting downstream target c-Myc gene transcription. Notably, our mechanistic study validated that HDAC7/β-catenin/c-Myc could form the positive feedback loop to enhance ESCC cell growth, and USP10 could deubiquitinate and stabilize HDAC7 protein in the ESCC cells. Additionally, we verified that inhibition of the HDAC7/β-catenin/c-Myc axis and USP10/HDAC7 pathway mediated the anti-proliferative action of melatonin on ESCC cells.

Conclusions: Our findings elucidate that melatonin mitigates the HDAC7/β-catenin/c-Myc positive feedback loop and inhibits the USP10-maintained HDAC7 protein stability thus suppressing ESCC cell growth, and provides the reference for identifying biomarkers and therapeutic targets for ESCC.

Keywords: Esophageal squamous cell carcinoma (ESCC); Histone deacetylase 7 (HDAC7); Melatonin; Ubiquitin-specific peptidase 10 (USP10); c-Myc; β-catenin.

Fig. 1

Melatonin induces cell proliferation inhibition, HDAC7 and c-Myc downregulation on ESCC cells. a Colony formation assay with melatonin pretreatments for 48 h. b Representative images and results of EdU incorporation assay. The result was calculated as the ratio between the number of EdU-stained cells (red fluorescence) and the total number of Hoechst 33342-stained cells (blue fluorescence). c Representative heatmap of HDACs gene expression based on the RNA‐seq results of EC109 cells. d Representative Western blotting results of HDACs. e Representative result of HDAC7 mRNA level. f Representative Western blotting results of c-Myc, p21, p27 and Cyclin D1. ^*P < 0.05 vs. the MEL 0 mmol/L group, ^#P < 0.05 vs. the MEL 1 mmol/L group, ^$P < 0.05 vs. the MEL 2 mmol/L group. HDAC7 histone deacetylase 7, ESCC esophageal squamous cell carcinoma, MEL melatonin

Fig. 2

High HDAC7 expression predicts poor prognosis of ESCC patients and melatonin inhibits ESCC proliferation via inhibiting HDAC7. a Representative IHC images for HDAC7 expression in ESCC tissues. Scale bar, 200 μm and 50 μm (inset), respectively. b Statistical analysis of HDAC7 expression in 148 ESCC patients through IHC staining. c Kaplan–Meier survival analysis by the high/low HDAC7 levels of the 148 ESCC patients based on the microarray tissue IHC results. d Representative HDAC7 protein level in different ESCC cell lines. e Photographs and results showing xenograft tumor morphologies and weights in each group after subcutaneously injection of NC or HDAC7 OE EC109 cells, respectively. f Representative images and results of colony formation and EdU incorporation assays. g Representative Western blotting results of HDAC7, c-Myc, p21, p27 and Cyclin D1. ^*P < 0.05 vs. the NC group or shCon group, ^#P < 0.05 vs. the MEL 4 mmol/L group, ^$P < 0.05 vs. the HDAC7 OE or shHDAC7 group. HDAC7 histone deacetylase 7, ESCC esophageal squamous cell carcinoma, MEL melatonin, OE overexpression, IHC immunohistochemistry

Fig. 3

HDAC7-c-Myc positive feedback loop to promote ESCC growth and involves in the anti-proliferative action of melatonin on ESCC cells. a Representative IHC images for c-Myc expression in ESCC tissues. Scale bar, 200 μm and 50 μm (inset), respectively. b Statistical analysis of c-Myc expression in 148 ESCC patients through IHC staining. c Kaplan–Meier survival analysis by high/low c-Myc levels of the 148 ESCC patients based on the microarray tissue IHC results. d Spearman correlation analyses of HDAC7 and c-Myc expression in the ESCC tissues. e Kaplan–Meier analysis of the association between overall survival and the expression of HDAC7 and c-Myc on 148 ESCC patients. f Representative Western blotting results of c-Myc, HDAC7, p21, p27 and Cyclin D1. g Representative Western blotting results of HDACs in the EC109 cells with c-Myc knockdown. h Representative Western blotting results of c-Myc, HDAC7, p21, p27 and Cyclin D1 in the EC109 and EC9706 cells with 10058-F4 treatment. i Representative images and results of colony formation and EdU incorporation assays. j Co-treatment of melatonin, LV-Flag-HDAC7 and LV-shc-Myc #1 was applied to EC109 cells. Representative Western blotting results of HDAC7, Flag-HDAC7, c-Myc, p21 and p27 were shown. ^*P < 0.05 vs. the NC group, ^#P < 0.05 vs. the HDAC7 OE group, ^&P < 0.05 vs. the MEL 4 mmol/L group, ^$P < 0.05 vs. the HDAC7 OE + MEL 4 mmol/L group. HDAC7 histone deacetylase 7, ESCC esophageal squamous cell carcinoma, OE overexpression, LV lentivirus, MEL melatonin, IHC immunohistochemistry

Fig. 4

HDAC7 promotes β-catenin deacetylation, dephosphorylation and nuclear transport, whereas melatonin interrupts above actions. a and b Co-IP analysis was performed to determine the interaction between HDAC7 and β-catenin in both EC109 and HEK-293T cells. HDAC7 or β-catenin proteins were respectively immunoprecipitated by the anti-Flag antibody followed by target proteins detection using Western blotting analyses. c Cytosolic fractionation and immunoblot assays were applied to detect β-catenin nuclear import after overexpressing HDAC7 by infecting LV-Flag-HDAC7 or melatonin treatment in EC109 cells; GAPDH and Lamin B1 were respectively cytoplasmic and nucleus loading controls. d and e Melatonin treatment for 48 h enhanced β-catenin phosphorylation (Ser675) and acetylation (Lys49) without affecting total β-catenin and TCF4 levels, whereas HDAC7 overexpression or downregulation could partially hinder or enhance the above melatonin’s actions. Representative Western blotting results of phosphorylated β-catenin (Ser675), acetylated β-catenin (Lys49), β-catenin and TCF4 were shown. HDAC7 histone deacetylase 7, LV lentivirus, MEL melatonin, TCF4 transcription factor 4, OE overexpression

Fig. 5

Lf3 and SAHA inhibits ESCC cell growth and β-catenin/c-Myc signaling, and melatonin co-treatment further enhances these actions. a and c Representative images and results of colony formation assay with Lf3 + melatonin or SAHA + melatonin pretreatments for 48 h in the EC109 and EC9706 cells. b and d Co-treatment of Lf3 + melatonin or SAHA + melatonin was applied to EC109 and EC9706 cells for 48 h. Representative Western blotting results of HDAC7, c-Myc, phosphorylated β-catenin (Ser675), acetylated β-catenin (Lys49), β-catenin, p21 and p27 were shown. ^*P < 0.05 vs. the Lf3 0 μmol/L group + MEL 0 mmol/L group, ^#P < 0.05 vs. the Lf3 0 μmol/L group + MEL 4 mmol/L group, ^$P < 0.05 vs. the only Lf3 treatment groups, ^&P < 0.05 vs. the only SAHA treatment groups. HDAC7 histone deacetylase 7, MEL melatonin

Fig. 6

USP10 deubiquitinates and stabilizes HDAC7 protein in ESCC cells. a Indicated Flag-tagged USPs and HA-HDAC7 were transfected into HEK-293T cells, and Flag-tagged HDAC7 was respectively transfected into HEK-293T and EC109 cells, then co-IP analysis was performed to determine the interaction between HDAC7 and USP10 in vitro by Western blotting analyses. b Immunoblot analyses of whole cell lyses derived from HEK-293T cells transfected with indicated Flag-USP10, HA-HDAC7 and GFP plasmids. c Immunoblot analyses of input and HDAC7 immunoprecipitated from EC109 cells with USP10 knocking down, and treated with 10 μmol/L MG132 overnight before harvesting. d Result of HDAC7 mRNA levels in the EC109 cells with USP10 knocking down. e HEK-293T cells were transfected with indicated Flag-USP10 and HA-HDAC7 plasmids, then cells were treated with CHX at indicated time points. The HDAC7 protein level was quantified by the ImageJ software and plotted. f Overview of USP10 protein structures. g HEK-293T cells transfected with the indicated constructs were subjected to pull down with anti-HA or anti-GST. h HDAC7 ubiquitylation was analyzed in HEK-293T cells transfected with USP10/WT or USP10/C424A. USPs ubiquitin-specific proteases, HDAC7 histone deacetylase 7, ub ubiquitin, EV empty vector, WT wild type, CHX cycloheximide, USP10 ubiquitin-specific peptidase 10

Fig. 7

Melatonin inhibits USP10-mediated HDAC7 deubiquitination thus inducing HDAC7 proteolysis. a Representative IHC images for USP10 expression in ESCC tissues. Scale bar, 200 μm and 50 μm (inset), respectively. b Statistical analysis of USP10 expression in 148 ESCC patients through IHC staining. c Kaplan–Meier survival analysis by high/low USP10 levels of the 148 ESCC patients based on the microarray tissue IHC results. d The spearman correlation analyses of USP10, HDAC7 and c-Myc expression in the ESCC tissues. e Kaplan–Meier analysis of the association between overall survival and the expression of USP10, HDAC7 and c-Myc in 148 ESCC patients. f Photographs and results showing xenograft tumor morphologies and weights in each group after subcutaneously injection of shCon or shUSP10 EC109 cells, respectively. g Representative images and results of colony formation and EdU incorporation assays in the EC109 cells with melatonin and USP10 knockdown treatment. h Western blotting results showed melatonin treatment for 48 h decreased USP10 levels in ESCC cells. i Immunoblot analyses of input and HDAC7 immunoprecipitated from EC109 cells with melatonin exposure and treated with 10 μmol/L MG132 overnight before harvesting. j Representative western blotting results of USP10 and HDAC7 in the EC109 cells with melatonin and USP10 knockdown treatment. ^*P < 0.05 vs. the shCon group, ^#P < 0.05 vs. the shUSP10 #1 group, ^&P < 0.05 vs. the USP10 #2 group, ^$P < 0.05 vs. the MEL 4 mmol/L group. USP10 ubiquitin-specific protease 10, HDAC7 histone deacetylase 7, ESCC esophageal squamous cell carcinoma, ub ubiquitin, MEL melatonin, LV lentivirus, IHC immunohistochemistry, USP10 ubiquitin-specific peptidase 10

Fig. 8

Schematic diagram about the molecular mechanism of melatonin inhibiting ESCC cell growth. ① USP10 deubiquitinates and stabilizes HDAC7 protein in the ESCC cells. Moreover, melatonin downregulates HDAC7 by both decreasing HDAC7 transcription and reducing protein stability through USP10 inhibition. ② HDAC7 physically deacetylates β-catenin Lys49, thus hindering β-catenin phosphorylation and subsequently activating target c-Myc gene expression. Interestingly, HDAC7/β-catenin/c-Myc could form a positive feedback loop to enhance ESCC cell growth, and melatonin works as a potent HDAC7 inhibitor thus prohibiting ESCC proliferation. USP10 ubiquitin-specific protease 10, HDAC7 histone deacetylase 7, ESCC esophageal squamous cell carcinoma