Inhibition of bromodomain-containing protein 4 enhances the migration of esophageal squamous cell carcinoma cells by inducing cell autophagy

Abstract

Background: Esophageal squamous cell carcinoma (ESCC), the predominant type of esophageal cancer, has a 5-year survival rate less than 20%. Although the cause of poor prognosis is the high incidence and mortality of ESCC, the high rate of metastasis after esophageal cancer surgery is the main cause of death after the surgery. Bromodomain-containing protein 4 (BRD4), an epigenetic reader of chromatin-acetylated histones in tumorigenesis and development, plays an essential role in regulating oncogene expression. BRD4 inhibition and BRD4 inhibition-based treatment can potentially suppress ESCC growth. However, the effects and mechanisms of action of BRD4 on ESCC cell migration remain unclear.

Aim: To explore the effect of BRD4 on cell migration of ESCC in vitro and its possible molecular mechanism.

Methods: Human ESCC cell lines KYSE-450 and KYSE-150 were used. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was performed to examine cell proliferation, and the transwell migration assay was conducted to test ESCC cell migration. JQ1, a BRD4 inhibitor, was applied to cells, and BRD4 siRNA was transfected into ESCC cells to knockdown endogenous BRD4. GFP-RFP-LC3 adenovirus was infected into ESCC cells to evaluate the effect of JQ1 on autophagy. Western blotting was performed to determine the protein levels of BRD4, E-cadherin, vimentin, AMP-activated protein kinase (AMPK), and p-AMPK.

Results: BRD4 was either downregulated by small interfering RNA or pretreated with JQ1 in ESCC cells, leading to increased tumor migration in ESCC cells in a dose- and time-dependent manner. Inhibition of BRD4 not only significantly suppressed cell proliferation but also strongly increased cell migration by inducing epithelial-mesenchymal transition (EMT). The protein expression of vimentin was increased and E-cadherin decreased in a dose-dependent manner, subsequently promoting autophagy in KYSE-450 and KYSE-150 cells. Pretreatment with JQ1, a BRD4 inhibitor, inhibited BRD4-induced LC3-II activation and upregulated AMPK phosphorylation in a dose-dependent manner. Additionally, an increased number of autophagosomes and autolysosomes were observed in JQ1-treated ESCC cells. The autophagy inhibitor 3-methyladenine (3-MA) reversed the effects of BRD4 knockdown on ESCC cell migration and blocked JQ1-induced cell migration. 3-MA also downregulated the expression of vimentin and upregulation E-cadherin.

Conclusion: BRD4 inhibition enhances cell migration by inducing EMT and autophagy in ESCC cells via the AMPK-modified pathway. Thus, the facilitating role on ESCC cell migration should be considered for BRD4 inhibitor clinical application to ESCC patients.

Keywords: Bromodomain-containing protein 4; Cell autophagy; Cell migration; Epithelial-mesenchymal transition; Esophageal squamous cell carcinoma; JQ1.

Figure 1

Effects of JQ1, an inhibitor of bromodomain-containing protein 4, on esophageal squamous cell carcinoma cell proliferation and migration. A: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide assay was conducted to detect cell viability of KYSE-450 cells and KYSE-150 cells after treatment with JQ1 at various fixed dosages at different time points; B: Phase contrast images of esophageal squamous cell carcinoma cells treated with JQ1 were captured by a Nikon digital microscope. C: Transwell assay was performed to examine cell migration in KYSE-450 cells and KYSE-150 cells after treatment with JQ1 at various fixed dosages; D: Western blot was carried out to measure the expression levels of E-cadherin and vimentin in KYSE-450 cells and KYSE-150 cells after treatment with JQ1 at various fixed dosages. GAPDH was used as the protein loading control. ^aP < 0.05, ^bP < 0.01. Scale bars in (B) and (C) are 100 μm.

Figure 2

Knockdown of bromodomain-containing protein 4 promotes esophageal squamous cell carcinoma cell migration. A: Transwell assay was performed to detect cell migration in KYSE-450 and KYSE-150 after transfection with siBRD4; B: Western blot was conducted to measure the levels of E-cadherin and vimentin after transfection with siBRD4. GAPDH was used as the protein loading control. ^aP < 0.05, ^bP < 0.01. Scale bar in (A) is 100 μm.

Figure 3

JQ1 induces cell autophagy and activates AMP-activated protein kinase. A: Cells infected by the GFP-RFP-LC3 virus were treated with dimethyl sulfoxide or JQ1 dissolved in dimethyl sulfoxide (5 μmol/L) for 24 h, then fluorescence microscopy images were photographed. Bar = 20 μm; B: Cells were either untreated or treated with JQ1 at doses of 0, 5, or 10 μmol/L for 24 h. Western blots were performed to examine the levels of LC3-II, AMP-activated protein kinase (AMPK) and p-AMPK in KYSE-450 cells and KYSE-150 cells. GAPDH was used as the protein loading control.

Figure 4

Inhibition of autophagy blocks JQ1-caused cell migration and epithelial-mesenchymal transition. A: Transwell assay was performed to detect cell migration of KYSE-450 and KYSE-150 after treatment with JQ1 (5 μmol/L) along or combination with 3-methyladenine (3-MA) (1 mmol/L); B: Western blots were performed to measure the levels of LC3-II, E-cadherin, and vimentin in KYSE-450 cells and KYSE-150 cells after treatment of JQ1 along or combination with 3-MA. GAPDH was used as the protein loading control. ^bP < 0.01. Scale bar in (A) is 100 μm.

Figure 5

Schematic model of the effect of JQ1 or inhibition of bromodomain-containing protein 4 on cell migration in esophageal squamous cell carcinoma cells. JQ1 treatment or siRNA inhibition of bromodomain-containing protein 4 can lead to increased phosphorylation of AMP-activated protein kinase, resulting in upregulation of LC3-II protein level in order to activate autophagy. In addition, the activation of autophagy promotes the epithelial-mesenchymal transition process of esophageal squamous cell carcinoma cells, thus promoting the JQ1-induced migration of esophageal cancer cells. Finally, this pro-migration effect can be inhibited by the autophagy inhibitor 3-methyladenine. AMPK: AMP-activated protein kinase; TF: Transcription factor; BRD4: Bromodomain-containing protein 4; EMT: Epithelial-mesenchymal transition; ESCC: Esophageal squamous cell carcinoma; AC: Acetylation.