Chen YM, Yang WQ, Fan YY, Chen Z, Liu YZ, Zhao BS. Trichostatin A augments cell migration and epithelial-mesenchymal transition in esophageal squamous cell carcinoma through BRD4/c-Myc endoplasmic reticulum-stress pathway. World J Gastroenterol 2025; 31(11): 103449 [PMID: 40124272 DOI: 10.3748/wjg.v31.i11.103449]
Corresponding Author of This Article
Bao-Sheng Zhao, MD, Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, No. 88 Jiankang Road, Weihui 453100, Henan Province, China. drbszhao@xxmu.edu.cn
Research Domain of This Article
Gastroenterology & Hepatology
Article-Type of This Article
Basic Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
World J Gastroenterol. Mar 21, 2025; 31(11): 103449 Published online Mar 21, 2025. doi: 10.3748/wjg.v31.i11.103449
Trichostatin A augments cell migration and epithelial-mesenchymal transition in esophageal squamous cell carcinoma through BRD4/c-Myc endoplasmic reticulum-stress pathway
Yan-Min Chen, Wen-Qian Yang, Yu-Zhen Liu, Bao-Sheng Zhao, Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
Yan-Min Chen, Department of Oncology, The First Affiliated Hospital of Henan Polytechnic University, Jiaozuo 454000, Henan Province, China
Wen-Qian Yang, Yu-Zhen Liu, Bao-Sheng Zhao, Henan Medical Science Key Laboratory of Esophageal Cancer Metastasis Translational Medicine, Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
Wen-Qian Yang, Yu-Zhen Liu, Life Science Research Center, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
Ying-Ying Fan, Department of Gastroenterology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
Zhi Chen, Department of Anesthesiology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
Co-corresponding authors: Yu-Zhen Liu and Bao-Sheng Zhao.
Author contributions: All authors contributed to the study’s conception and design; Chen YM, Liu YZ, and Zhao BS made contributions to the conception and design of this study; Chen YM, Yang WQ and Chen Z conducted experiments; Fan YY, Yang WQ and Zhao BS were responsible for clinical sample collection; Chen Y and Yang WQ participated in data analysis; Chen YM, Liu YZ, Yang WQ and Zhao BS wrote the manuscript.
Supported by the Henan Province Science and Technology Development Plan, No. 242102311124; Key Medical Scientific and Technological Project of Henan Province, No. SBGJ202102188; Henan Provincial Medical Science and Technology Project, No. LHGJ20221012; and Fundamental Research Funds for the Universities of Henan Province, No. NSFRF240308.
Institutional review board statement: This study was reviewed and approved by the First Affiliated Hospital of Xinxiang Medical University Institutional Review Board (Approval No. EC-024-559).
Institutional animal care and use committee statement: This study does not involve any animal experimentation.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: No additional data are available.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Bao-Sheng Zhao, MD, Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, No. 88 Jiankang Road, Weihui 453100, Henan Province, China. drbszhao@xxmu.edu.cn
Received: November 25, 2024 Revised: January 9, 2025 Accepted: February 14, 2025 Published online: March 21, 2025 Processing time: 111 Days and 1.3 Hours
Abstract
BACKGROUND
The causes of death in patients with advanced esophageal cancer are multifactorial, with tumor metastasis being one of the important factors. Histone acetylation promotes the migration of esophageal squamous cell carcinoma (ESCC) cells, while the histone deacetylase inhibitor (HDACi) shows complex effects on tumor functions.
AIM
To comprehensively elucidate the impact and molecular mechanisms of trichostatin A (TSA), an HDACi, on cell migration in ESCC through bromodomain-containing protein (BRD4)/cellular myelocytomatosis oncogene (c-Myc)/endoplasmic reticulum (ER)-stress.
METHODS
The effects of TSA on ESCC cell lines Eca109 and EC9706 migration were evaluated using Transwell assays, with small interfering transfection and pathway-specific inhibitors to elucidate underlying mechanisms. The mRNA levels involved were examined by quantitative real-time polymerase chain reaction. Protein levels of acetylated histones H3 (acH3) and acetylated histones H4, BRD4, c-Myc, as well as markers of ER stress and epithelial-mesenchymal transition (EMT), were analyzed using western blot. Additionally, this method was also used to examine acH3 levels in esophageal cancer tissues and adjacent tissues. Patient outcomes were subsequently tracked to identify prognostic indicators using Log-Rank tests and Cox multivariate analysis.
RESULTS
TSA promoted the migration of ESCC cells by stimulating the EMT process. TSA-mediated histone acetylation facilitated the recruitment of BRD4, a bromodomain-containing protein, triggering the expression of c-Myc. This cascade induced ER stress and enhanced EMT in ESCC cells. To further elucidate the underlying mechanism, we employed various interventions including the ER stress inhibitor 4-phenylbutyric acid, knockdown of c-Myc and BRD4 expression, and utilization of the BRD4 inhibitor carboxylic acid as well as the inhibitor of TSA 1. Mechanistically, these studies revealed that TSA-mediated histone acetylation facilitated the recruitment of BRD4, which in turn triggered the expression of c-Myc. This sequential activation induced ER stress and subsequently enhanced EMT, thereby promoting the migration of ESCC cells. Additionally, we examined histone acetylation levels in specimens from 43 patients with ESCC, including both tumor tissues and paired adjacent tissues. Statistical analysis unveiled a negative correlation between the level of histone acetylation and the long-term prognosis of patients with ESCC.
CONCLUSION
TSA promoted ESCC cell migration through the BRD4/c-Myc/ER stress pathway. Moreover, elevated histone acetylation in ESCC tissues correlated with poor ESCC prognosis. These findings enhance our understanding of ESCC migration and HDACi therapy.
Core Tip: This study revealed that trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), promoted the migration of esophageal squamous cell carcinoma (ESCC) cells by facilitating epithelial-mesenchymal transition (EMT) through the bromodomain-containing protein (BRD4)/cellular myelocytomatosis oncogene (c-Myc)/endoplasmic reticulum (ER) stress pathway. Mechanistically, TSA-induced histone acetylation enhances BRD4 recruitment and c-Myc expression, which triggers ER stress and subsequently drives EMT. Clinical analyses demonstrated a negative correlation between histone acetylation levels and ESCC prognosis, providing novel insights into the molecular mechanisms of ESCC migration and the implications of HDACi therapy.
Citation: Chen YM, Yang WQ, Fan YY, Chen Z, Liu YZ, Zhao BS. Trichostatin A augments cell migration and epithelial-mesenchymal transition in esophageal squamous cell carcinoma through BRD4/c-Myc endoplasmic reticulum-stress pathway. World J Gastroenterol 2025; 31(11): 103449
Globally, esophageal cancer ranks as the eighth most prevalent cancer and holds the sixth position in terms of cancer-related deaths[1]. It is predominantly classified into two histological types: Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma[2]. In China, ESCC accounts for over 90% of esophageal cancer[3]. Despite advancements in cytoreductive surgery and chemotherapy, the five-year survival rate for ESCC remains under 20%, largely due to factors such as metastasis, recurrence, and resistance to chemotherapy[4]. This underscores the critical need to investigate new therapeutic agents and molecular markers that can predict outcomes, aiming to enhance treatment effectiveness for patients with ESCC.
Histone acetylation, a crucial epigenetic modification, regulates gene transcription and influences biological processes like cell survival and migration[5]. This modification is tightly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs)[6]. While histone acetylation by HATs activates gene transcription by loosening chromatin structure, HDACs counteract this process by removing acetyl groups, leading to chromatin compaction and gene transcriptional silencing. The dysregulation of histone acetylation, often attributed to aberrant HDACs activity, is frequently observed in various cancers and correlates with tumorigenesis and poor prognosis[7]. Therefore, inhibiting HDACs activity presents a potential therapeutic strategy in oncology.
Histone deacetylase inhibitors (HDACi) have emerged as novel anti-tumor agents by specifically inhibiting HDACs enzymes, thereby preventing histone deacetylation and elevating histone acetylation levels[8]. This promotes gene transcription, particularly of tumor suppressor genes, exerting an anti-tumor effect[9]. Among these, romidixin, parbital, berilastat, and vorinostat (SAHA) stand out as HDACi sanctioned by the Food and Drug Administration (FDA) for the management of conditions like cutaneous T-cell lymphoma, peripheral T-cell lymphoma, and multiple myeloma[10]. This highlights the clinical relevance and therapeutic potential of targeting HDACs activity in cancer management. However, recent clinical trials have shown that the efficacy of HDACi in treating solid tumors does not meet expectations. Some studies have suggested that the dysregulation of the dynamic balance between acetylation and deacetylation may play a dual role in certain tumors[11]. Elevated acetylation levels can effectively inhibit tumor cell proliferation, but may also pose inherent risks[12]. Specifically, heightened acetylation could induce the overexpression of oncogenes that facilitate tumor cell metastasis[13]. HDACi have been implicated in promoting cell migration and metastasis by activating genes associated with tumor progression across various cancer types, including liver, colon, and lung cancers[14-16]. Consequently, there is an urgent need to investigate the mechanisms through which HDACi induce migration and invasion of ESCC tumor cells. Such research is crucial for the development of comprehensive treatment strategies involving HDACi.
In recent years, studies investigating the potential mechanism of HDACi in promoting tumor metastasis have focused on epithelial-mesenchymal transition (EMT), a crucial process involved in cancer cell migration and invasion[17]. These investigations have received significant attention. For instance, SAHA has been found to acetylate the transcription factor Snail, thereby promoting its transcription and nuclear translocation, ultimately enhancing the invasiveness of hepatocellular carcinoma cells[18]. Similarly, valproic acid has been shown to induce EMT in breast cancer cells by upregulating the transcription of ZEB1, consequently enhancing their migratory capabilities[19].
During the process of tumor cascade, it often leads to misfolding protein reactions causing endoplasmic reticulum (ER) stress[20]. EMT and ER stress can mutually activate one another[21]. For example, agents like Chlamydia trachomatis and carotenoids have been shown to induce EMT-like phenotypes in thyroid cells by inducing ER stress[22]. Similarly, during EMT, cancer cells change the secretion of extracellular viral matrix protein, thereby promoting ER stress[23]. Furthermore, it has been reported that colorectal cancer cells can induce the EMT process to activate the unfolded protein response (UPR) pathway under stable overexpression of hypoxia inducible factor-1α or serum starvation[24]. These results highlight the complex interplay between HDACi, EMT, and cancer metastasis. Therefore, a better understanding of the interplay between HDACi, EMT, and ER stress is essential.
We have previously shown that trichostatin A (TSA) enhances ESCC cell migration through EMT via histone acetylation and acetylating RelA at K310[25,26]. These findings suggest that TSA facilitates EMT in ESCC cells via distinct regulatory pathways. Therefore, investigating other potential molecular mechanisms through which TSA promotes ESCC cell migration could aid in developing more effective strategies for combined HDACi treatment in patients with ESCC. In this study, we investigated the impact of ER stress signaling on TSA-induced EMT and cell migration in ESCC cells. We discovered that TSA triggered ER stress through bromodomain-containing protein (BRD4)/cellular myelocytomatosis oncogene (c-Myc) signaling, subsequently promoting EMT and cell migration in ESCC cells. Additionally, our analysis revealed that histone acetylation levels associated with poor prognosis in patients with esophageal cancer.
MATERIALS AND METHODS
Cells and cell culture
Human ESCC cell lines EC9706 and Eca109 were purchased from Cobioer Biosciences (Nanjing, Jiangsu Province, China). These cells were cultured in dulbecco’s modified eagle medium (Corning Costar, Manassas, VA, United States) with 10% fetal bovine serum (Gibco, Waltham, MA, United States) in a humidified atmosphere of 5% carbon dioxide at 37 °C.
Human tissue specimens collected from ESCC patients
Tissue specimens were collected from 43 patients diagnosed with ESCC at The First Affiliated Hospital of Xinxiang Medical University between April 1, 2017 and January 31, 2018. Collected specimens comprised both neoplastic tissue and adjacent esophageal mucosa, situated approximately 5 cm away from the edge of the tumor. Importantly, each participant in the study had not received radiotherapy or chemotherapy prior to surgery and had given informed consent. Additionally, the study protocol was approved by the Ethics Committee of The First Affiliated Hospital of Xinxiang Medical University.
Cell morphological observation
EC9706 and Eca109 cells were seeded in six-well plates (4 × 105 cells/well of each). After overnight incubation, the cells were treated with TSA (Beyotime Biotechnology, Shanghai, China, S1893) for a duration of 24 hours. Subsequently, cell morphology was examined using a phase-contrast microscope (Nikon, TS2, Japan), and the images were captured utilizing NIS-Elements imaging system (Nikon, TS2, Japan).
Transwell migration assay
Cell migration was examined using a Transwell assay. Briefly, 200 μL of EC9706 or Eca109 cell suspension (1 × 105 cells/mL in serum-free medium) was added into the upper chamber of a 6.5-mm Transwell insert with an 8-μm pore membrane (Corning Costar, Manassas, United States) and 600 μL of complete medium was filled into the lower chamber. Various compounds, TSA, 4-phenylbutyric acid (4-PBA) (an ER stress inhibitor) (Sigma, St. Louis, MO, United States, SML0309), JQ-1 (Beyotime Biotechnology, Shanghai, China, SC0002), or inhibitor of TSA 1 (ITSA1) (Selleck, Houston, United States, S8323), were applied to both chambers at indicated doses. The cells were allowed to migrate for 24 hours, after which non-migratory cells on the upper membrane surface were removed with a cotton swab. The cells that had migrated to the lower surface were then fixed using 4% paraformaldehyde and stained with 0.1% crystal violet. Images from five random fields per membrane were taken with a phase-contrast microscope (Nikon, TS2, Japan), and the migrated cells were quantified.
Small interfering RNA transfection
EC9706 and Eca109 cells, at a density of 4 × 105, were cultured overnight in six-well plates. When the cells were 80% confluence, the medium was replaced by 1.8 mL of basic medium. Then, 2 μg of small interfering (si)-BRD4, sic-Myc or si-negative control (GenePharma, Shanghai, China) was separately mixed with 100 μL of Opti-MEM (Gibco, Thermal Fisher Scientific, Waltham, MA, United States) essential medium, while 2 μL of lipofectamine 2000 (Invitrogen, Waltham, Massachusetts, United States) was mixed with 100 μL of Opti-MEM basic medium. After 10 minutes, the two solutions were separately added to each well and thoroughly mixed with the cells. Following 28 hours of culturing, the transfected cells were harvested for subsequent experiments. The small interfering RNA sequences were as follows: C-Myc: 5’-GCTTGTACCTGCAGGATCT-3’, BRD4: 5’-GACACUAUGAAACACCAGTT-3’, and negative control: 5’-GCGACCAACGCCTTGATTG-3’.
Western blot analysis
Cells were lysed with radioimmunoprecipitation assay lysis buffer. Equal amounts of protein extracts (30 μg) were separated to sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, United States). Membranes were initially blocked with 5% nonfat dry milk and subsequently incubated overnight at 4 °C with specific primary antibodies: acetylated histones H3 (acH3) (CST, 4353), H3 (CST, 4499), acetylated histones H4 (acH4) (Abcam, ab177790), H4 (Abcam, ab177840), N-cadherin (CST, 13116), E-cadherin (CST, 3195), vimentin (CST, 5741), β-catenin (CST, 8480), activating transcription factor 6 (ATF6) (Wanleibio, WL01153), C/EBP homologous protein (CHOP) (Wanleibio, WL00880), glucose-regulated protein (GRP) 78 (Wanleibio, WL03157), c-Myc (CST, 13987), and β-actin (CST, 4967) (all diluted 1:1000). Following washing, the membranes were then treated with horseradish peroxidase-linked secondary antibodies. After incubation, the membranes were washed three more times with phosphate-buffered saline and then exposed to SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, United States). Finally, the blots were examined using the AmershamTM Imager 600 System (GE Healthcare BioSciences, Pittsburgh, United States).
Statistical analysis
Statistical evaluations were performed using statistical product and service solutions version 27.0. Categorical variables were presented as frequencies or percentages and were analyzed using the χ2 test. For normally distributed continuous data, values were presented as means ± SD, with comparisons between groups conducted via independent sample t-tests. Survival curves were generated using Kaplan-Meier survival analysis, and the impact of ac-H3 expression levels on patient prognosis in ESCC was evaluated using the Log Rank test.
RESULTS
TSA promotes migration of ESCC cells by facilitating EMT
Increasing evidence suggests that elevated histone acetylation promotes tumor metastasis[25]. In this study, consistent with our previous findings in other ESCC cell lines[26,27], TSA promoted the migration of Eca109 and EC9706 cells in a dose-dependent manner (Figure 1A and B), accompanied by a morphological change in Eca109 and EC9706 cells from their original oval epithelioid morphology to a fusiform stromal morphology (Figure 1C). We then speculated whether this phenomenon was related to EMT. To explore this, western blot experiments were conducted and, as expected, TSA enhanced the levels of acH3 and acH4. Additionally, the mRNA and protein levels of E-cadherin were decreased, while vimentin and β-catenin were increased (Supplementary Figure 1, Figure 1D and E) in ESCC cells treated with different concentrations of TSA. These findings suggest that TSA facilitates ESCC cell migration via EMT.
Figure 1 Trichostatin A promotes migration of esophageal squamous cell carcinoma cells by promoting epithelial-mesenchymal transition.
A: Representative images of the migration of Eca109 and EC9706 cells treated with histone deacetylase inhibitor trichostatin A (TSA) from Transwell assays; B: TSA enhanced cell migration in Transwell migration assay in dose-dependent manners; C: Changes in Eca109 and EC9706 cell morphology after TSA treatment; D and E: Western blot shows after TSA treatment that acetylated histones H3 and acetylated histones H4 levels increased and epithelial-mesenchymal transition marker E-cadherin level decreased, vimentin, β-catenin levels increased. Cell counts are for the corresponding assays of at least five random microscope fields (100 magnification). n = 5, P vs control. aP < 0.05. bP < 0.01. Panel A Bar is 50 μm, panel B Bar is 10 μm. TSA: Trichostatin A; acH3: Acetylated histones H3; acH4: Acetylated histones H4.
TSA promotes EMT in ESCC cells by inducing ER stress
HDACi elevate intracellular histone acetylation levels, boosting protein synthesis[28]. This increased protein synthesis can lead to ER stress, triggering an adaptive response within the ER[29]. Studies have linked ER stress to tumor metastasis regulation[30], prompting speculation about the role of TSA in promoting EMT in ESCC cells via ER stress. Thus, we examined the protein levels of ER stress markers, including GRP78, CHOP, and ATF6. As shown in Figure 2A, TSA increased these protein levels in a dose-dependent manner. Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) results showed that TSA elevated the mRNA levels of GRP78, CHOP, and ATF6 in ESCC cells (Supplementary Figure 2A-C). Furthermore, 4-PBA, an ER stress inhibitor, attenuated TSA-induced ESCC cell migration (Figure 2B and Supplementary Figure 2D). Western blot analysis revealed that 4-PBA reduced the TSA-induced elevation of GRP78 levels and shifted the protein expression profile induced by TSA towards mesenchymal-epithelial transition (Figure 2C). These findings strongly suggest that TSA promotes ESCC cell migration through ER stress.
Figure 2 Trichostatin A promotes epithelial-mesenchymal transition of esophageal squamous cell carcinoma cells by promoting endoplasmic reticulum stress.
A: Western blot shows after trichostatin A (TSA) treatment, that endoplasmic reticulum (ER) stress marker glucose-regulated protein 78, C/EBP homologous protein and activating transcription factor 6 levels increased. These show that TSA can promote ER stress in esophageal cancer cells; B: Representative images of the migration of Eca109 and EC9706 cells treated with TSA combined with 4-phenylbutyric acid (4-PBA) from Transwell assays. The statistical results show that TSA enhanced cell migration reversed by 4-PBA in the Transwell migration assay; C: Western blot shows after TSA combined with 4-PBA, the epithelial-mesenchymal transition marker E-cadherin level increased, vimentin, β-catenin levels deceased. Cell counts are for the corresponding assays of at least five random microscope fields (100 magnification). n = 5, P vs control. aP < 0.05. bP < 0.01. Bar is 50 μm. TSA: Trichostatin A; 4-PBA: 4-phenylbutyric acid; GRP78: Glucose-regulated protein 78; CHOP: C/EBP homologous protein; ATF6: Activating transcription factor 6.
TSA promotes EMT in ESCC cells through c-Myc-mediated ER stress
TSA increases histone acetylation levels in cells, thereby modulating the expression of associated genes. Among these genes, c-Myc, a potent oncogene regulated by histone acetylation[31], plays a crucial role in various malignancies, including solid tumors and leukemia[32]. Activation of c-Myc has been reported to induce ER stress, leading to corresponding biological effects[33]. To explore whether TSA-induced ER stress in ESCC cells is mediated by c-Myc, Western blot and RT-qPCR were conducted, revealing an increase in c-Myc protein and mRNA levels following TSA treatment of ESCC cells (Figure 3A and Supplementary Figure 3A). The knockdown of c-Myc using siRNA in ESCC cells (Figure 3B and Supplementary Figure 3B) significantly reversed TSA-induced ESCC cell migration (Figure 3C). Further western blot analysis revealed that knockdown of c-Myc reversed the expression of ER stress marker protein GRP78 and EMT-related proteins to a certain extent (Figure 3D). Collectively, these results suggest that TSA promotes ESCC cell migration by upregulating c-Myc, thereby triggering ER stress and EMT.
Figure 3 Trichostatin A promotes epithelial-mesenchymal transition of esophageal squamous cell carcinoma cells through cellular myelocytomatosis oncogene mediated endoplasmic reticulum stress.
A: Western blot shows after trichostatin A (TSA) treatment that cellular myelocytomatosis oncogene (c-Myc) level increased; B: Relative protein level of c-Myc in control cells and c-Myc-knockdown cells treated with TSA was examined by western blot; C: Representative images of the migration of Eca109 and EC9706 cells knockdown c-Myc protein and treated with TSA examined by Transwell assays; D: Western blot shows after TSA combined with sic-Myc that endoplasmic reticulum stress and epithelial-mesenchymal transition marker E-cadherin level increased, glucose-regulated protein 78, vimentin, β-catenin level decreased. Cell counts are for the corresponding assays of at least five random microscope fields (100 magnification). n = 5, P vs control. aP < 0.05. bP < 0.01. Bar is 50 μm. TSA: Trichostatin A; GRP78: Glucose-regulated protein 78; c-Myc: Cellular myelocytomatosis oncogene.
TSA-promoted EMT in ESCC cells through c-Myc-mediated ER stress may be associated with BRD4
Our previous studies demonstrated that TSA promotes the migration of ESCC cell lines KYSE-150 and EC9706 through BRD4, an essential epigenetic “reader” protein that recognizes acetylated lysine. BRD4 is known for its role in regulating transcriptional elongation factors and key genes associated with cell cycle progression and apoptosis, including c-Myc and Bcl2[34,35]. As expected, TSA increased histone H3 acetylation (Figure 2D) in ESCC cells. Therefore, we speculated that TSA-induced histone H3 acetylation recruits BRD4, which then activates c-Myc to induce ER stress, subsequently promoting EMT. Interestingly, TSA increased the expression of BRD4 mRNA and protein (Figure 4A and Supplementary Figure 4). To further verify the role of BRD4, we used the BRD4 inhibitor carboxylic acid (JQ-1), and knocked down BRD4 expression via transcription of siBRD4 (Figure 4B, Supplementary Figure 4). Transwell experiments showed that both JQ-1 treatment and BRD4 knockdown reversed the TSA-induced promotion of ESCC cell migration (Figure 4C and D). Western blot revealed that BRD4 knockdown inhibited TSA-induced upregulation of c-Myc as well as ER-stress marker GRP78 levels, and at least partially reversed EMT-related protein levels (Figure 4E). We conclude that TSA promotes ESCC cell migration by recruiting BRD4, which then activates c-Myc to induce ER stress, subsequently promoting EMT.
Figure 4 Trichostatin A promotes epithelial-mesenchymal transition of esophageal squamous cell carcinoma cells through cellular myelocytomatosis oncogene mediated endoplasmic reticulum stress may be associated with bromodomain-containing protein.
A: Western blot shows after trichostatin A (TSA) treatment that bromodomain-containing protein (BRD4) level increased; B: Relative protein level of BRD4 in control cells and BRD4-knockdown cells treated with TSA was examined by western blot; C: Representative images of the migration of Eca109 and EC9706 cells treated with TSA combined with carboxylic acid from Transwell assays; D: Representative images of the migration of Eca109 and EC9706 cells treated with TSA combined with knocking down BRD4 from Transwell assays; E: Western blot shows TSA treated of knock down BRD4 esophageal squamous cell carcinoma cells that epithelial-mesenchymal transition marker E-cadherin level increased, vimentin, β-catenin level decreased. Cell counts are for the corresponding assays of at least five random microscope fields (100 magnification). n = 5, P vs control. aP < 0.05. bP < 0.01. Bar is 50 μm. TSA: Trichostatin A; JQ-1: Carboxylic acid; GRP78: Glucose-regulated protein 78; BRD4: Bromodomain-containing protein.
TSA-promoted EMT in ESCC cells through the BRD4/c-Myc/ER-stress pathway is reversed by ITSA1
In our study, we observed that TSA promotes the migration of ESCC cells through the BRD4/c-Myc/ER stress pathway. To validate this mechanism, we used the ITSA1, which counteracts TSA-induced histone acetylation and transcriptional activation. Our findings showed that ITSA1 not only reversed the heightened histone acetylation levels induced by TSA but also alleviated the migratory effect of TSA on ESCC cells (Figure 5A and Supplementary Figure 5). Additionally, ITSA1 reversed the expression levels of proteins associated with the c-Myc/ER stress signaling pathway (Figure 5B). These results collectively confirmed that TSA-induced migration in ESCC cells occurs via EMT triggered through the histone acetylation-induced c-Myc/ER stress pathway.
Figure 5 Trichostatin A promotes epithelial-mesenchymal transition of esophageal squamous cell carcinoma cells through bromodomain-containing protein/cellular myelocytomatosis oncogene/endoplasmic reticulum-stress pathway is reversed by inhibitor of trichostatin A 1.
A: Representative images of the migration of Eca109 and EC9706 cells treated with trichostatin A (TSA) combined with inhibitor of trichostatin A 1 (ITSA1) from Transwell assays; B: Western blot shows after TSA combined with ITSA1 that acetylated histones H3, acetylated histones H4, cellular myelocytomatosis oncogene, glucose-regulated protein 78 levels decreased, and epithelial-mesenchymal transition marker E-cadherin levels increased, vimentin, β-catenin levels decreased. Cell counts are for the corresponding assays of at least five random microscope fields (100 magnification). aP < 0.05. bP < 0.01. Bar is 50 μm. TSA: Trichostatin A; ITSA1: Inhibitor of trichostatin A 1; GRP78: Glucose-regulated protein 78; acH3: Acetylated histones H3; c-Myc: Cellular myelocytomatosis oncogene.
Histone acetylation correlates with the clinical progression of ESCC
In this study, we demonstrated that elevated levels of histone acetylation promote the migration of ESCC cells. To explore the involvement of histone acetylation in ESCC development and progression within clinical contexts, we examined both cancerous and adjacent non-cancerous tissues obtained from 43 patients with ESCC. Detailed clinical and pathological characteristics of these patients are provided in the Supplementary Table 1. Western blot analysis was employed to assess acH3 levels, and the representative results are shown in Figure 6A. Quantitative analysis revealed that acH3 levels ESCC tumor tissues were 1.66 times higher than those in adjacent tissues (Figure 6B). Kaplan-Meier survival analysis was conducted with 43 patients during the follow-up period. AcH3 levels were categorized as high or low based on whether they were above or below the median, respectively. Significant differences in overall survival time were observed between the low and high acH3 level groups, as determined by the Log-Rank test (Figure 6C, P = 0.029). Univariate analysis revealed associations between the acH3 level, tumor differentiation, and survival outcome (Table 1, P < 0.1). Cox multivariate survival analysis identified a high acH3 level rather than differentiation as an independent risk factor for survival (Table 2, P < 0.05, hazard ratio = 1.950, 95% confidence interval: 1.001-3.804). These findings underscore the prognostic significance of histone H3 acetylation in esophageal cancer, indicating that higher levels of histone acetylation correlate with poorer prognosis. Collectively, these clinical data are consistent with our previous in vitro results on cell migration initiated by TSA.
Figure 6 Expression of acetylated histones H3 is higher in esophageal squamous cell carcinoma than in paracancerous tissues, and acH3 was an independent factor affecting prognosis.
A: Representative detection of acetylated histones H3 (acH3) level in esophageal squamous cell carcinoma (ESCC) by western blot, N means adjacent tissues and C means cancer tissues; B: Statistical analysis of 43 cases of ESCC showed that the level of acH3 in ESCC tumor tissue was 1.66 times higher than that in adjacent tissue; C: Kaplan-Meier survival curve of the relationship between acH3 expression and prognosis in patients with esophageal carcinoma. P vs control. bP < 0.01. NC: Negative control; acH3: Acetylated histones H3.
Table 1 Univariate analysis of patients’ survival outcome.
Factors
P value
HR
95%CI
Lower limit
Upper limit
Gender
0.231
1.532
0.763
3.075
Age
0.852
0.933
0.447
1.945
Tumor location
0.995
0.998
0.517
1.928
Tumor max
0.235
1.567
0.712
2.876
Differentiation
0.096
0.677
0.427
1.072
Depth of infiltration
0.5448
0.891
0.610
1.300
Lymphatic metastasis
0.903
0.960
0.495
1.863
TNM staging
0.621
0.831
0.400
1.727
High blood pressure
0.581
1.230
0.589
2.570
acH3 expression
0.040
2.012
1.033
3.920
Table 2 Analysis of multiple factors influencing survival outcome of patients.
Factors
P value
HR
95%CI
Lower limit
Upper limit
Differentiation
0.122
0.684
0.424
1.106
acH3 expression
0.043
1.980
1.101
3.704
DISCUSSION
Since the FDA’s approval of SAHA for cutaneous T-cell lymphoma in 2006, HDACi have emerged as promising candidates for cancer treatment[36]. While HDACi generally demonstrate inhibitory effects on cell proliferation across various cancer types[37-40], emerging research suggests that specific HDACi like SAHA and TSA paradoxically facilitate migration and invasion. This phenomenon is attributed to their ability to induce transcription factors such as ZEB1, ZEB2, and slug, which promote EMT in prostate and colorectal cancer cells[41,42]. In this study, we further demonstrated that TSA enhances migration in ESCC cells through the induction of EMT, despite its inhibitory effect on ESCC proliferation (Supplementary Figure 6). This migratory response is mediated by the activation of ER stress.
ER stress arises from disturbances in protein folding within the ER, leading to the activation of the UPR, a cellular signaling pathway aimed at restoring ER homeostasis[43]. ER stress has been linked to various aspects of cancer biology, including cell proliferation[44,45], apoptosis[46], and metastasis[47]. Emerging evidence suggests a link between ER stress and EMT in cancer progression. ER stress signaling pathways have been implicated in the regulation of EMT-associated transcription factors such as snail, slug, and twist, as well as EMT-related signaling pathways including transforming growth factor-β and Wnt/β-catenin[21,48]. ER stress reduces E-cadherin and Mucin-16 expression while increasing vimentin and snail levels, indicating a shift from epithelial to mesenchymal traits in thyroid cells[49]. Under hypoxic conditions, alveolar epithelial cells experiencing ER stress exhibit heightened expression of mesenchymal markers both in vivo and in vitro[46]. Inhibition of ER stress can reduce the vitality, migration, and invasiveness of triple-negative breast cancer cells by regulating the syntenin/Sox4/Wnt/β-catenin pathway via HSPA4[50]. In hepatocellular carcinoma cells, the hepatitis B virus surface antigen triggers ER stress, boosting fibroblast growth factor 19 production. This activates the Janus kinase/STAT3 pathway, inducing EMT[51]. Despite advancements, understanding of HDACi-induced ER stress in metastasis remains limited. Therefore, further investigation into the specific impact of TSA-induced ER stress on ESCC cell migration is warranted. In this study, we demonstrated for the first time that TSA induces ER stress, thereby enhancing ESCC cell migration through EMT. We subsequently elucidated the specific mechanism by which TSA-induced ER stress promotes ESCC cell migration, highlighting the potential role of ER stress in promoting aggressive tumor phenotypes in ESCC.
The increased production of misfolded proteins is a critical factor in ER stress[43]. c-Myc, a crucial nuclear transcription factor, plays a significant role in tumorigenesis and progression[52]. Studies have shown that c-Myc enhances protein synthesis by facilitating ribosomal biogenesis and mRNA translational efficiency[53]. Moreover, c-Myc boosts cellular metabolic activity and oxidative stress levels, potentially leading to or exacerbating ER stress[54]. The regulation of c-Myc expression by histone HDACi appears to be multifaceted and contingent upon the cellular context. While some HDACi, such as SAHA and MS-275, have been observed to significantly upregulate c-Myc expression in certain cancer cell lines, others like valproic acid exhibit contrasting effects, leading to a reduction in c-Myc expression[55,56]. These findings underscore the intricate nature of HDACi-mediated modulation of c-Myc levels, suggesting differential mechanisms underlying the regulation of c-Myc expression by different HDACi. In this study, we found that TSA significantly elevated c-Myc expression in ESCC cells, providing further insight into the complex interplay between HDACi and c-Myc in cancer cells.
Histone acetylation, typically associated with transcriptional activation, serves as a pivotal epigenetic modification regulating gene expression[57]. During this process, “reader” proteins, such as bromodomain-containing proteins like BRD4, play a crucial role[58]. These proteins bind to acetylated histones, recruiting transcriptional machinery and chromatin remodelers to gene promoters[59]. c-Myc regulation by HDACi is complex and involves various mechanisms, including alterations in histone acetylation patterns and the recruitment of transcriptional regulators like BRD4[60]. Previous research has shown that BRD4 activates c-Myc expression by forming a complex with the c-Myc promoter region[32]. Conversely, BRD4 inhibition has been shown to decrease c-Myc levels in certain tumors[61,62]. In this study, we extensively investigated the impact of BRD4 on c-Myc expression and its contribution to promoting EMT in ESCC cells under ER stress induced by TSA.
Our study observed an upregulation of BRD4 in ESCC cells following TSA treatment. Notably, inhibiting TSA-induced histone acetylation abrogated this upregulation, suggesting that TSA-induced histone acetylation facilitates the transcription of the BRD4. However, we currently lack direct evidence demonstrating the binding of acetylated histones, such as acH3, to the BRD4 promoter. Literature sources indicate that BRD4 transcription is regulated by various transcription factors, including c-Myc, nuclear factor kappa-B, activator protein-1, GATA family members, and E2F[63]. The specific transcription factors involved can vary depending on the cell type and physiological state. Histone acetylation, as an epigenetic modification, is known to relax chromatin structure, thereby enhancing the accessibility of multiple gene promoters to transcription factors.
Considering these factors, we hypothesize that the upregulation of BRD4 mRNA induced by TSA, potentially via histone acetylation, may also involve multiple transcription factors. To validate this hypothesis, further studies, such as chromatin immunoprecipitation, are needed to complete the investigation. Despite this, our study provides the first evidence of the pivotal role of the BRD4/c-Myc/ER stress pathway in TSA-induced EMT in ESCC cells.
EMT is a critical mechanism driving cell migration and plays an significant role in tumor progression[64]. During EMT, there is an upregulation of mesenchymal markers, such as vimentin, alongside a downregulation of epithelial markers, including E-cadherin. β-catenin, a key component of Wnt signaling pathway, can translocate to the nucleus and act as a transcriptional co-activator for genes that promote mesenchymal characteristics[65]. Acetylation of histones H3 and H4, a key epigenetic modification, regulates gene expression. In this study, the application of ITSA1 significantly blocked the TSA-induced elevation of acH3 and acH4 subsequent to the activation of the BRD4/c-Myc/ER stress cascade, and consequently, EMT. These findings suggest that TSA-induced alterations in histone acetylation within the chromatin landscape are associated with the EMT process.
Research findings indicate that histone acetylation plays a significant role in tumor progression, showing varying patterns across different cancer types. The level of histone acetylation is regulated by many factors, including the homeostasis of HATs and HDACs. Studies have found that the expression of HDACs is closely related to the prognosis of patients with esophageal cancer[66], and high expression of histone deacetylases 6 and histone deacetylases 7 are associated with poor prognosis[67,68]. Increased histone acetylation levels have been associated with a higher risk of progression in patients with ESCC stages IIB and III[69]. Our study specifically demonstrated an upregulation of histone acetylation levels in ESCC tissues. Notably, patients with ESCC and high acH3 levels displayed unfavorable overall survival times. These findings underscore the importance of considering histone acetylation levels, in addition to HDACs levels, when evaluating the use of HDACi in patients with ESCC. This result underscore the need for a personalized treatment approach with HDACi tailored to the histone acetylation status of individual patients with ESCC, potentially leading to improved therapeutic outcomes.
Our study demonstrated that TSA enhances histone acetylation levels in ESCC cells, consequently promoting BRD4-mediated upregulation of c-Myc expression and activating ER stress-induced EMT (Figure 7). However, further experimental evidence is required to fully understand the precise regulatory mechanisms underlying histone acetylation, BRD4, c-Myc, ER stress, and EMT.
Figure 7 A proposed model illustrating the mechanism by which trichostatin A promotes esophageal squamous cell carcinoma cell epithelial-mesenchymal transition and migration.
ac: Acetylation; TSA: Trichostatin A; HAT: Histone lysine acetyltransferase; HDAC: Histone deacetylases; ER: Endoplasmic reticulum; EMT: Epithelial-mesenchymal transition; c-Myc: Cellular myelocytomatosis oncogene.
CONCLUSION
In summary, our research demonstrated that TSA promotes the migration of human ESCC cells through EMT, involving the BRD4/c-Myc/ER-stress signaling cascade. Additionally, we observed an elevation in histone acetylation in ESCC cancer tissues, correlating with an unfavorable prognosis, indicating its potential as a prognostic biomarker. These findings advance our understanding of the molecular mechanisms underlying ESCC migration with HDACi and underscore the importance of considering both histone acetylation and HDACs levels in evaluating HDACi use for patients with ESCC.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade B, Grade C, Grade C
Novelty: Grade B, Grade B, Grade B
Creativity or Innovation: Grade B, Grade B, Grade B
Scientific Significance: Grade B, Grade B, Grade B
P-Reviewer: Li MJ; Xu BT S-Editor: Fan M L-Editor: A P-Editor: Zhang L
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