Histone deacetylase inhibitors promote epithelial-mesenchymal transition in Hepatocellular Carcinoma via AMPK-FOXO1-ULK1 signaling axis-mediated autophagy

Abstract

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related deaths globally because of high metastasis and recurrence rates. Elucidating the molecular mechanisms of HCC recurrence and metastasis and developing effective targeted therapies are expected to improve patient survival. The promising anti-cancer agents for the treatment of hematological malignancies, histone deacetylase inhibitors (HDIs), have limited effects against epithelial cell-derived cancers, including HCC, the mechanisms involved have not been elucidated. Herein, we studied the molecular mechanisms underlying HDI-induced epithelial-mesenchymal transition (EMT) involving FOXO1-mediated autophagy. Methods: The biological functions of HDIs in combination with autophagy inhibitors were examined both in vitro and in vivo. Cell autophagy was assessed using the generation of mRFP-GFP-LC3-expressing cells and fluorescent LC3 puncta analysis, Western blotting, and electron microscopy. An orthotopic hepatoma model was established in mice for the in vivo experiments. Results: Our study provided novel mechanistic insights into HDI-induced EMT mediated by the autophagy AMPK-FOXO1-ULK1-Snail signaling axis. We demonstrated that autophagy served as a pro-metastasis mechanism in HDI-treated hepatoma cells. HDIs induced autophagy via a FOXO1-dependent pathway, and FOXO1 inhibition promoted HDI-mediated apoptosis in hepatoma cells. Thus, our findings provided novel insights into the molecular mechanisms underlying HDI-induced EMT involving FOXO1-mediated autophagy and demonstrated that a FOXO1 inhibitor exerted a synergistic effect with an HDI to inhibit cell growth and metastasis in vitro and in vivo. Conclusion: We demonstrated that HDIs triggers FOXO1-dependent autophagy, which ultimately promotes EMT, limiting the clinical outcome of HDI-based therapies. Our study suggests that the combination of an HDI and a FOXO1 inhibitor is an effective therapeutic strategy for the treatment of HCC.

Keywords: Autophagy; Epithelial-mesenchymal transition; FOXO1 inhibitor; Histone deacetylase inhibitors; Metastasis.

Figure 1

HDI-induced EMT, invasion, and metastasis in hepatoma cells. (A) Cells were incubated in the presence of different concentrations of HDIs for 24 h, and the cytotoxicity of HDIs in hepatoma cells was determined by the MTT enzyme assay. (B-E) HepG2 and PLC cells were treated with or without 3 mM NaB (dissolved in water) or 3 µM SAHA (dissolved in DMSO) for 24 h, and phenotypic changes of EMT were detected using a phase-contrast microscope (B). Protein expression of vimentin, fibronectin, and Snail was detected by Western blotting (C). Invasion and metastasis of hepatoma cells were detected by wound-healing and cell invasion assays, respectively (D-E). Scale bar, 200 µm. Similar results were obtained in three independent experiments.

Figure 2

HDI-induced autophagy in hepatoma cells. (A) HepG2 and PLC cells were treated with or without 3 mM NaB or 3 µM SAHA for 6, 12, and 24 h, and markers of autophagy (LC3-I, LC3-II, P62) were detected by Western blotting. (B) The mRFP-eGFP-LC3 plasmid was transfected into HepG2 and PLC cells for 24 h, and the cells were treated with or without 3 mM NaB or 3 µM SAHA for 12 h; fluorescent were counted under confocal microscopy, scale bar, 5 µm. Similar results were obtained in three independent experiments. ***P<0.0001.

Figure 3

Role of FOXO1 and ULK1 in regulating autophagy and EMT in hepatoma cells. (A-B) HepG2 and PLC cells were treated with or without 3 mM NaB or 3 µM SAHA for 6 h; the expression of autophagy-related genes was assessed using gene chip technology (A), mRNA levels of FOXO1 and ULK1 were detected by real-time PCR (B). (C) HepG2 and PLC cells were treated with or without 3 mM NaB or 3 µM SAHA for 6 h, 12 h, and 24 h, and the protein expression levels of FOXO1 and ULK1 were detected by Western blotting. Similar results were obtained in three independent experiments. (D-G) HepG2 and PLC cells were transfected with or without FOXO1 siRNA (D-E) or ULK1 siRNA (F-G) for 24 h; the cells were treated with or without 3 mM NaB or 3 µM SAHA for 24 h, and the expression levels of Snail, vimentin, LC3I, LC3II and P62 were detected by Western blotting. Similar results were obtained in three independent experiments.

Figure 4

FOXO1 inhibitor antagonizes HDI-induced autophagy and EMT in hepatoma cells. (A) HepG2 and PLC cells were incubated in the presence of different concentrations of the FOXO1 inhibitor AS for 24 h, and the cytotoxicity of AS in hepatoma cells was determined by the MTT assay. (B) HepG2 and PLC cells were pretreated with or without 1 µM AS for 24 h and then treated with or without 3 mM NaB or 3 µM SAHA for 24 h, and the protein expression levels of FOXO1, ULK1, LC3-I, LC3-II, Snail, and vimentin were detected by Western blotting. (C-E) HepG2 and PLC cells were pretreated with or without 1 µM AS for 24 h and then with or without 3 mM NaB or 3 µM SAHA for different times; the morphological structure of autophagy were examined by transmission electron microscopy, scale bar, 1 µm (C) Invasion and metastasis were detected by wound-healing assay (D) and (E) cell invasion assay. Similar results were obtained in three independent experiments.

Figure 5

FOXO1 inhibitor represses HDI-induced expression of Snail by inhibiting Smad2/3 phosphorylation and nuclear translocation. (A) Cells were pretreated with or without 1 µM AS for 24 h followed by treatment with or without 3 mM NaB or 3 µM SAHA for 15, 30 min, and 1 h. The phosphorylation of ULK1 and Smad2/3 was detected by Western blotting. GAPDH served as the loading control. Similar results were obtained in three independent experiments. (B) Cells were pretreated with or without 1 µM AS for 24 h and then with or without 3 mM NaB or 3 µM SAHA for 30 min, and immunofluorescence and confocal microscopy were used to detect Smad2/3, scale bar, 20 µm. (C) Cells were pretreated with or without 1 µM AS for 24 h and then with or without 3 mM NaB or 3 µM SAHA for 1 h, and the binding of Smads to the Snail promoter was detected by EMSA. (D) Cells were transfected with pGL3-Basic-Snail-luc reporter plasmid, pretreated with or without 1 µM AS for 24 h, and then with or without 3 mM NaB or 3 µM SAHA for 24 h. Luminescence was measured using a luminometer. pRL-TK plasmids served to correct for transfection efficiency. The results are expressed as the ratios between the activity of the reporter plasmid and pRL-TK, *p < 0.01. (E) Cells were pretreated with or without 1 µM AS for 24 h and then with or without 3 mM NaB or 3 µM SAHA for 24 h. Snail mRNA was detected by qRT-PCR.

Figure 6

FOXO1 inhibitor combined with HDIs inhibits proliferation and promotes apoptosis in hepatoma cells. (A) HepG2 and PLC cells were treated with NaB or SAHA and AS at indicated concentrations. Cell viability was determined by the MTT enzyme assay, *p < 0.01. (B-C) HepG2 and PLC cells were pretreated with or without 1 µM AS for 24 h and then with or without 3 mM NaB or 3 µM SAHA for 24 h; (B) cell apoptosis was calculated by flow cytometry, (C) Expression of Cleaved PARP and Cleaved Caspase-3 was measured by Western blotting. Similar results were obtained in three independent experiments.

Figure 7

HDIs upregulate FOXO1 by activating AMPK and inhibiting the degradation of FOXO1. (A) HepG2 and PLC cells were treated with or without 3 mM NaB or 3 µM SAHA for 10, 15, 30, 45, and 60 min. AMPK phosphorylation was detected by Western blotting. GAPDH served as a loading control. Similar results were obtained in three independent experiments. (B) Cells were pretreated with or without 4 µM of the AMPK inhibitor Compound C (Cc) for 2 h, then treated with or without 3 mM NaB or 3 µM SAHA for 12 h, and the expression of FOXO1, ULK1 and Snail was detected by qRT-PCR and Western blotting. GAPDH served as the loading control. Similar results were obtained in three independent experiments. (C) Cells were treated with or without 3 mM NaB or 3 µM SAHA for 1, 2, and 4 h, and FOXO1 expression was detected by Western blotting. GAPDH served as the loading control. Subsequently, cells were treated with or without 3 mM NaB or 3 µM SAHA for 2 h, and the total proteins in the cell lysate were subjected to immunoprecipitation with an anti-FOXO1 antibody. The acetylation or ubiquitination of FOXO1 levels in the immune complex was detected by Western blotting with an anti-acetylate or an anti-ubiquitin antibody; FOXO1 served as the loading control. Similar results were obtained in three independent experiments. (D) A proposed model to illustrate the mechanism of HDI induction of FOXO1 expression and the promotion of hepatoma cell EMT and autophagy. →direct stimulation; ─|direct inhibition.

Figure 8

HDIs combined with a FOXO1 inhibitor elicit a protective antitumor response in HepG2 tumor-bearing mice. (A) Schematic representation of the treatment schedule and dosages in mice. (B-D) 5×10⁶ HepG2-luc cells were injected into the livers of nude mice with a 1 mL insulin injection needle, and the wounds were sutured with 5-0 silk thread. After 14 days, the mice were assigned to the following six groups, Saline, SAHA, NaB, AS, AS+SAHA, and AS+NaB. Mouse survival (B) and body weights (C) were measured at regular intervals; (D) The tumor volumes and metastasis were detected by in vivo imaging using luciferin. Representative images of metastatic liver tumors are shown.