Inhibition of BRD4 promotes migration of esophageal squamous cell carcinoma cells with low ACC1 expression

doi:10.12122/j.issn.1673-4254.2025.10.22

Abstract

Abstract:

Objective To investigate the effect of BRD4 inhibition on migration of esophageal squamous cell carcinoma (ESCC) cells with low acetyl-CoA carboxylase 1 (ACC1) expression. Methods ESCC cell lines with lentivirus-mediated ACC1 knockdown or transfected with a negative control sequence (shNC) were treated with DMSO, JQ1 (a BRD4 inhibitor), co-transfection with shNC-siBRD4 or siNC with additional DMSO or C646 (an ahistone acetyltransferase inhibitor) treatment, or JQ1combined with 3-MA (an autophagy inhibitor). BRD4 mRNA expression in the cells was detected using RT-qPCR. The changes in cell proliferation, migration, autophagy, and epithelial-mesenchymal transition (EMT) were examined with CCK8 assay, Transwell migration assay, and Western blotting. Results ACC1 knockdown did not significantly affect BRD4 expression in the cells but obviously increased their sensitivity to JQ1. JQ1 treatment at 1 and 2 μmol/L significantly inhibited ESCC cell proliferation, while JQ1 at 0.2 and 2 μmol/L promoted cell migration. The cells with ACC1 knockdown and JQ1 treatment showed increased expresisons of vimentin and Slug and decreased expression of E-cadherin. BRD4 knockdown promoted migration of ESCC cells, and co-transfection with shACC1 and siBRD4 resulted in increased vimentin and Slug expressions and decreased E-cadherin expression in the cells. C646 treatment of the co-transfected cells reduced acetylation levels, decreased vimentin and Slug expressions, and increased E-cadherin expression. Treatment with JQ1 alone obviously increased LC3A/B-II levels in the cells either with or without ACC1 knockdown. In the cells with ACC1 knockdown and JQ1 treatment, additional 3-MA treatment significantly decreased the expressions of vimentin, Slug and LC3A/B-II and increased the expression of E-cadherin. Conclusion BRD4 inhibition promotes autophagy of ESCC cells via a histone acetylation-dependent mechanism, thereby enhancing EMT and ultimately increasing cell migration driven by ACC1 deficiency.

Key words: esophageal squamous cell carcinoma, acetyl-CoA carboxylase 1, BRD4, migration

Wenxin JIA, Shuhua HUO, Jiaping TANG, Yuzhen LIU, Baosheng ZHAO. Inhibition of BRD4 promotes migration of esophageal squamous cell carcinoma cells with low ACC1 expression[J]. Journal of Southern Medical University, 2025, 45(10): 2258-2269.

Figures/Tables 9

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence (5'-3')
ACC1-F ACC1-R	ATCCCGTACCTTCTTCTACTG
ACC1-F ACC1-R	CCCAAACATAAGCCTTCACTG
BRD4-F BRD4-R	TGGATGCCGTCAAGCTGAAC
BRD4-F BRD4-R	GTTTCTTCTGTGGGTAGCTCATT
GAPDH-F GAPDH-R	AATGGGCAGCCGTTAGGAAA
GAPDH-F GAPDH-R	GCGCCCAATACGACCAAATC

Fig.1 Expression levels of BRD4 mRNA and protein in esophageal squamous cell carcinoma (ESCC) cells with and without ACC1 knockdown. A: RT-qPCR for detecting BRD4 mRNA level. B: Western blotting for determining the protein expression level of BRD4. ****P<0.0001.

Fig.2 BRD4 inhibitors antagonize the proliferation of ESCC cells. A: Effect of JQ1 on proliferation of KYSE-150/KYSE-450 (shNC, shACC1) cells detected by CCK-8 assay. B: CCK-8 assay for assessing the effect of different concentrations of JQ1 on proliferation of ESCC cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.3 The promotion of ESCC cell migration by BRD4 inhibitors. Transwell migration assay for evaluating the effect of different concentrations of JQ1 on migration of ESCC cells (Original magnification: ×100). **P<0.01, ****P<0.0001.

Fig.4 Inhibition of BRD4 promotes EMT in ESCC cells. The results of the Western blotting experiment showed that JQ1 inhibited the expression of E-cadherin protein and promoted the expression of vimentin and Slug. *P<0.05, ***P<0.001, ****P<0.0001.

Fig.5 Knockdown of BRD4 promotes migration of ESCC cells. A: Transwell migration assay for assessing the effect of BRD4 knockdown on migration of ESCC cells (×100). B: Western blotting for detecting expression levels of BRD4 and EMT-related proteins. **P<0.01, ***P<0.001, ****P<0.0001.

Fig.6 Histone acetyltransferase inhibitors antagonize migration of shACC1 cells after BRD4 knockdown. A: Transwell migration assay for assessing the effect of C646 on migration of ESCC cells with both ACC1 and BRD4 knockdown (×100). B: Western blotting for detecting expression levels of BRD4 and EMT-related proteins in the cells after treatment with C646. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.7 Inhibition of BRD4 promotes migration of ESCC cells with low ACC1 expression through autophagy. A: Western blotting showing that 2 μmol/L JQ1 promotes expressions of LC3A/BII in KYSE-150 and KYSE-450 cells. B: 4 mmol/L 3-MA inhibits migration of the cells with ACC1 knockdown and JQ1 treatment (×100). ***P<0.001, ****P<0.0001.

Fig.8 Inhibition of autophagy counteracts the effect of BRD4 inhibition on EMT of ESCC cells with low expression of ACC1. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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