AGS GC cell line (CRL-1739) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). AGS cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland) and 1× penicillin/streptomycin (Lonza). All experiments were performed after AGS cells reached the exponential growth phase.

A small-molecule inhibitor of the BET family, iBET-151, was purchased from Selleckchem (Houston, TX, USA) and prepared as a 10-mM stock solution in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Paclitaxel, an anti-mitotic drug, was purchased from Sigma-Aldrich.

For differential expression analysis by RNA-seq, total RNA was isolated from iBET-151- or/and paclitaxel-treated or untreated AGS cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA quantity and integrity were assessed using both Nanodrop and Bioanalyzer, according to the manufacturer’s instructions. Library preparation and RNA-seq was performed using the Illumina TruSeq RNA Library Preparation Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Samples were sequenced on the Illumina NovaSeq 6000, 150 bp paired-end reads, to a minimum depth of 120 million reads per sample. Sequenced reads were aligned to the hg19 genome assembly using TopHat2. Differential expression analysis was processed following the Tuxedo protocol. DEGs were defined based on the cutoff values of p ≤ 0.05, false discovery rate < 0.05, and fold change (FC) > 1.5. The reference annotation based on Ensembl release 69 (ftp://ftp.ensembl.org/pub/release-69/gtf/macaca_mulatta).

GO annotation enrichment analysis was performed using The Database for Annotation, Visualization, and Integrated Discovery (DAVID) (v6.8), an online tool applied for functional annotation analysis and Gene Set Enrichment Analysis using MSigDB. GO and KEGG pathway analyses provide a comprehensive set of functional annotation tools for identifying characteristic biological attributes of high-throughput genome or transcriptome data.

To study the effect of drugs on protein expression, tissues and cells were lysed in RIPA buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 10% glycerol) containing protease and phosphatase inhibitors (Roche Diagnostics, Laval, QC, Canada). The cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. Membranes were then developed using an enhanced chemiluminescence (ECL) reagent (GE Healthcare, Piscataway, NJ, USA). α-tubulin was used as a loading control. Antibodies used for western blotting included anti‒epidermal growth factor receptor (EGFR), anti‒fibroblast growth factor receptor (FGFR)-3, and anti‒insulin-like growth factor-1 receptor (IGF-1R; Cell Signaling Technology, Beverly, MA, USA); anti‒human epidermal growth factor receptor (HER)-2, anti‒HER-3, and anti-MET (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-α-tubulin (Sigma-Aldrich); and horseradish peroxidase-conjugated secondary anti-rabbit and anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) antibodies. Membranes were then developed using the ECL reagent (GE Healthcare), and α-tubulin was used as a loading control. Quantification was performed with a Molecular Imager ChemiDoc XRS+ Imaging System (Bio-Rad, Hercules, CA, USA)

Graphing was performed using GraphPad Prism software version 8 (Graph Pad Software Inc., San Diego, CA, USA). All statistical analyses were performed using SPSS version 25 (IBM Corp., Armonk, NY, USA). All the significance levels were set at p ≤ 0.05, and all p-values were two-sided.

In our previous study, we confirmed that iBET-151-paclitaxel combination treatment exerted a synergistic effect on cell viability and invasion in several cell lines (unpublished data). To understand the effects of iBET-151 and paclitaxel on the whole transcriptome better, we performed RNA-seq of AGS cells that were most affected by the synergistic effects of iBET-151 and paclitaxel to compare the global gene expression profiles of untreated and iBET-151-, paclitaxel-, and iBET-151‒paclitaxel combination-treated cells.

About 16,000 genes were selected after the preprocessing step, during which genes that were not detected in both treated and untreated AGS cells were excluded. Based on our DEG-specific criteria, we observed that iBET-151 upregulated 239 genes, whereas paclitaxel upregulated 203 genes (Fig. 1A). Upregulation of 270 genes by combination treatment was mainly due to the effect of iBET-151 treatment (Fig. 1A). Of the genes upregulated by iBET-151 alone, 61% (167/270) were upregulated by the combination of iBET-151 and paclitaxel. Thus, these genes were shared between the combination- and iBET-151‒treated cells.

Then, we observed that iBET-151 downregulated 395 genes, whereas paclitaxel downregulated 57 genes (Fig. 1B). In the combination-treated cells, 358 genes were downregulated; among them, 73% (263/358) overlapped with the downregulated genes in the iBET-151‒treated cells (Fig. 1B). However, the genes regulated by paclitaxel alone did not overlap with those regulated by iBET-151 alone or the iBET-151-paclitaxel combination. The top 10 genes upregulated and downregulated by iBET-151, paclitaxel, and combination treatments are shown in Tables 1 and 2, respectively.

To determine the biological effects of gene expression changes, we performed GO analysis with the selected DEGs using DAVID v6.8. Volcano plots were used for the concurrent demonstration of both FC and p-value of DEGs in mono- or combination-treated cells (Figs. 2A, 3A, and 4A).

Interestingly, GO analysis showed that upregulated genes in iBET-151‒treated cells were most enriched in the chromatin silencing, nucleosome assembly pathways, and DNA packing complex (Fig. 2B, Supplementary Fig. 1A). This was because iBET-151 suppressed the epigenetic regulatory function of the BET family and affected chromatin remodeling. The genes downregulated by iBET-151 were enriched in angiogenesis; cell migration; RAS protein signal transduction; positive regulation of extracellular signal-regulated kinase (ERK)-1 and ERK cascade; positive regulation of transcription, DNA-templated; and negative regulation of apoptotic process (Fig. 2C, Supplementary Fig. 1B).

In paclitaxel-treated cells, the upregulated genes were enriched in the apoptotic signaling pathway; release of cytochrome c from mitochondria; regulation of apoptotic process; and mitochondrial electron transport, cytochrome c to oxygen (Fig. 3B, Supplementary Fig. 1C). Genes downregulated by paclitaxel were enriched in sister chromatid segregation, mitotic nuclear division, and centrosome duplication (Fig. 3C, Supplementary Fig. 1D). According to a recent study [16], paclitaxel inhibited mitosis by binding to microtubules and regulating mitosis-related pathways, including mitotic nuclear division, chromosome segregation, and G2/M transition of the mitotic cell cycle, in lung cancer cells. The results of our previous study on lung cancer cells showed that paclitaxel upregulated mitosis-related pathways, including mitotic nuclear division and chromosome segregation (unpublished data).

GO analysis showed a significant increase in the upregulation of the same genes involved in the aforementioned pathways in combination-treated cells. Moreover, an increase in the upregulation of genes involved in reactive oxygen species metabolic process and nucleotide-excision repair, DNA damage recognition was observed after combination treatment (Fig. 4B, Supplementary Fig. 1E). We observed that downregulated genes in the combination- and iBET-151-treated cells were enriched in overlapping GO terms, including angiogenesis, positive regulation of RAS protein signaling, cell migration, MAP kinase activity, and positive regulation of signaling; however, a significant decrease in gene expression was observed between the combination- and mono-treated cells (Fig. 4C, Supplementary Fig. 1F).

To understand the effects of combination treatment better, we identified genes that were more significantly upregulated or downregulated in combination-treated cells than in mono-treated cells. We first identified the genes with FC > 1.5 and p < 0.05 in combination-treated cells, compared with in control cells. We then identified the DEGs with FC > 1.5 in the respective mono-treated cells.

We identified 460 DEGs, including 238 upregulated and 222 downregulated DEGs, in AGS cells based on the above-mentioned cutoff values. As shown in Fig. 5A, the heatmap of hierarchical clustering of 460 DEGs demonstrated clear discriminating patterns. Then, GO analysis demonstrated that combination treatment upregulated genes enriched in nucleosome assembly, mitochondrial electron transport, and microtubule-based process (Fig. 5B). Subsequent GO analysis after combination treatment showed downregulation of genes involved in mitotic sister chromatid segregation, positive regulation of transcription, positive regulation of GTPase activity, and mitotic nuclear division (Fig. 5C).

Most of these results after combination treatment were similar to those obtained after iBET-151 or paclitaxel mono-treatment. However, combination treatment induced more changes in the genes than those in case of either of the mono-treatments.

Next, we determined the mRNA expression levels of BET protein target genes. We observed that iBET-151 reduced the mRNA expression of the oncogenes, MYC and KRAS, and the anti-apoptotic gene, BCL-2. However, no change was observed in the expression of these genes on paclitaxel treatment, as expected (Fig. 6A).

In our previous study, we found that iBET-151 induced G1 cell cycle arrest (unpublished data). Consistent with this result, the expression of cyclin dependent kinase-4/6 and E2F transcription factor-2 involved in G1-S phase transition was reduced. To elucidate the underlying biology, we observed the changes in major receptor tyrosine kinase (RTK) expression. We confirmed that iBET-151 reduced the expression of RTK mRNA and proteins, including EGFR, ERBB3, MET, and IGF-1R, thereby regulating cancer cell growth and survival. Additionally, RTK expression was further reduced on iBET-151‒paclitaxel combination treatment. However, in the FGFR family, FGFR-3/4 was significantly increased on iBET-151 and combination treatments (Fig. 6B and 6C).