Super-enhancers (SE) are clusters of transcription enhancers that drive gene expression. SEs are typically characterized by high levels of acetylation of histone H3 lysine 27 (H3K27ac), which is catalyzed by the histone lysine acetyltransferase CREB binding protein (CBP). Cancer cells frequently acquire tumor-specific SEs at key oncogenes, such as MYC, which induce several hallmarks of cancer. BRD4 is recruited to SEs and consequently functions as an epigenetic reader to promote transcription of SE-marked genes in cancer cells. miRNAs can be potent candidates for nucleic acid therapeutics for cancer. We previously identified miR-766-5p as a miRNA that downregulated MYC expression and inhibited cancer cell growth in vitro. In this study, we show that miR-766-5p directly targets CBP and BRD4. Concurrent suppression of CBP and BRD4 cooperatively downregulated MYC expression in cancer cells but not in normal cells. Chromatin immunoprecipitation analysis revealed that miR-766-5p reduced levels of H3K27ac at MYC SEs via CBP suppression. Moreover, miR-766-5p suppressed expression of a BRD4-NUT fusion protein that drives NUT midline carcinoma. In vivo administration of miR-766-5p suppressed tumor growth in two xenograft models. Collectively, these data suggest that targeting SEs using miR-766-5p–based therapeutics may serve as an effective strategy for the treatment of MYC-driven cancers.

Significance:

This study demonstrates that miR-766-5p targets CBP and BRD4, which can mitigate the protumorigenic consequences of SEs and oncogenic fusion proteins.

Super-enhancers (SE) are clusters of transcriptional enhancers that drive the expression of cell-type–specific genes (1, 2). SEs are typically characterized by high levels of acetylation of histone H3 lysine 27 (H3K27Ac; refs. 1, 2), which is catalyzed by the highly homologous histone lysine acetyltransferases CREB binding protein (CBP) and p300 (3, 4). Cancer cells frequently acquire tumor-specific SEs at key oncogenes, such as MYC, which induces several hallmarks of cancer (1, 5). MYC regulates autonomous proliferation, DNA replication, and cellular metabolism, and suppresses antitumor immune response, thereby promoting tumorigenesis (6–8). BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, is recruited to SEs and consequently functions as an epigenetic reader to promote the transcription of MYC in cancer cells (9, 10). Importantly, these SEs at the MYC locus are observed in many types of cancer cells, but not in noncancer cells (1). Thus, targeting SEs may be rational for cancer therapeutics.

Aberrant activation of WNT/β-catenin signaling featuring nuclear accumulation of β-catenin due to CTNNB1 or APC mutation is observed in many types of cancers (11). Nuclear β-catenin forms a complex with TCF/LEF transcription factors, upregulating the expression of MYC (12). A recent study revealed that WNT/β-catenin signaling also cooperates with SEs to increase the expression of MYC via cancer cell–specific gating of MYC in cancer cells (13). WNT/β-catenin–induced MYC upregulation reduces the sensitivity of acute myeloid leukemia and colon cancer cells to BET inhibitors, thus the combination of a BET inhibitor with the inhibition of WNT/β-catenin signaling is effective for inhibiting tumor growth (14–16).

miRNAs, small noncoding RNAs of about 22 nucleotides in length, function as endogenous gene repressors by binding to the complementary mRNA sequences of their target genes (17). miRNAs can be potent candidates of nucleic acid therapeutics for cancer, which are currently under development, especially when they target multiple genes that exert synergistic antitumor effects (18). We previously defined the growth-inhibitory effects of 2,565 miRNAs in HCT116 p53+/+ (HCT116+/+) and HCT116 p53−/− (HCT116−/−) cells, and extracted 138 miRNAs that markedly inhibited the tumor growth in vitro in both cell lines (19). We screened the effects of these 138 miRNAs on MYC suppression using a MYC reporter assay in HCT116−/− cells, as HCT116 cells have increased MYC expression through tumor-specific SEs (1, 5). We identified miR-766-5p as a miRNA that suppressed SEs and WNT/β-catenin signaling by targeting CREBBP (which encodes CBP), BRD4, and FRAT2. The therapeutic potential of miR-766-5p was demonstrated by local administration of miR-766-5p in a mouse xenograft model.

Cell culture

The cell lines used in this study are summarized in Supplementary Table S1. HCT116-derived isogenic p53-null cell line (HCT116−/−) was gifted from Dr. Bert Vogelstein (The Johns Hopkins University, Baltimore, MD). Ty82-JQ1R cells were previously generated from Ty82 cells (20). KYSE150 and RT7 cells were gifted from Dr. Y. Shimada (Toyama University, Toyama, Japan) and Dr. N. Kamata (Hiroshima University, Higashihiroshima, Japan), respectively (21, 22). All cell lines, which were maintained at 37°C and 5% CO2, were authenticated by monitoring cell morphology. Cells were tested for Mycoplasma contamination using the TaKaRa PCR Mycoplasma Detection Set (TaKaRa) and were cultured for no more than 20 passages from the validated stocks. We conducted all experiments according to the approved guidelines and regulations.

Antibodies

The primary antibodies used in this study are summarized in Supplementary Table S2.

Reagents

PKF118–310 and JQ1 were purchased from Cayman Chemicals and Selleck Chemicals, respectively. These reagents were used for the treatment of cultured cells in vitro at the indicated concentrations.

Transfection of miRNAs and siRNAs

The double-stranded RNAs (dsRNA) mimicking mature human miR-766-5p (MC23847) and a nonspecific control miRNA (negative control #1) were obtained from Thermo Fisher Scientific. The SMARTpool siRNA for BRD4 (M-004937–02), CREBBP (M-003477–02), FRAT2 (M-012406–01), MYC (M-003282–07), and nonspecific control siRNAs were purchased from Horizon Discovery. Each SMARTpool siRNA consists of four distinct siRNA duplexes targeting different mRNA sequences of the same gene. si-BRD4 (M-004937–02) downregulated the BRD4-NUT fusion gene by targeting coding regions of BRD4, as reported previously (20). miRNAs and siRNAs were transfected into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer's instructions.

MYC reporter assay

MYC reporter activity was assessed using Cignal Myc Reporter Assay Kit (luc) (CCS-012L; QIAGEN) according to the manufacturer's instructions. Six hours after transfection of HCT116−/− cells with the MYC E-box-luciferase reporter plasmid using Lipofectamine 2000, each of the 138 dsRNAs selected from the miRVana miR mimic Library v21 (Thermo Fisher Scientific) or miR-NC was added in duplicate using an RNA concentration of 10 nmol/L. Forty-eight hours later, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). To calculate the relative luciferase activity, Firefly luciferase activity was normalized to its corresponding internal Renilla luciferase control. MYC reporter activity was assessed using a relative ratio compared with that of miR-NC–transfected cells. All experiments were performed twice.

Quantitative RT-PCR

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The primers were designed using Primer3 plus (https://primer3plus.com/) based on sequence data obtained from NCBI databases (http://www.ncbi.nlm.nih.gov/). The endogenous control for mRNA was GAPDH. The primers used in this study are listed in Supplementary Table S3. For miRNA detection, total RNA was reverse transcribed using the TaqMan Reverse Transcription Kit followed by qRT-PCR performed using Custom TaqMan miRNA assays (Applied Biosystems). The expression level of miRNA was normalized to that of the internal control RNU6B. The following primers were used for the TaqMan assay (Thermo Fisher Scientific): human miR-766-5p (472145_mat) and RNU6B (001093). RNA of normal tissues (bladder, breast, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, prostate, spleen, stomach, and testis) were purchased from Biochain Institute Inc.

In vitro cell growth assay

Cell survival was evaluated by the crystal violet staining assay as described previously (19).

Gene expression array analysis

Gene expression array analysis was carried out as described previously (20). Briefly, HCT116−/− and MIAPaCa2 cells were transfected with 10 nmol/L miR-NC or miR-766-5p, and total RNA was extracted 48 hours after transfection. According to the manufacturer's instructions (Agilent Technologies), the Agilent 8 × 60 K array was used for gene expression analysis. The data were analyzed by GeneSpring software (Agilent Technologies). The data were deposited in the Gene Expression Omnibus (GEO; accession number GSE180688).

Western blotting

Western blotting was performed as described previously (20). β-Actin or vinculin was used as the loading control.

Glycolysis assay

To evaluate glycolysis, lactate production in the cultured cells was assessed using the Glycolysis Cell-Based Assay Kit (Cayman Chemical) according to the manufacturer's instructions. Briefly, 24 hours after transfection of HCT116−/− and MIAPaCa2 cells with miR-NC or miR-766-5p, si-NC, or si-MYC, culture medium was replaced with that containing 1% FBS to avoid high background. Eight hours later, the l-lactate in supernatant was measured and each value was normalized by the cell number. All experiments were performed in triplicate.

TCF/LEF reporter assay

TCF/LEF reporter assay was performed using the Cignal T-cell factor (TCF)/lymphoid enhancer factor (LEF) Reporter (luc) Kit (CCS-018L; QIAGEN) as described previously (23). Briefly, 2 days after cotransfection of Cignal Reporter or Cignal Negative Control as the internal control and miR-NC, miR-766-5p, si-NC, or indicated siRNAs into the indicated cells, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assay was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer's instructions. Briefly, 1 × 106 cells were harvested 48 hours after transfection with siRNA or miRNA, and then treated with 1% formaldehyde for 10 minutes at room temperature to cross-link proteins to DNA. Cross-linked DNA was lysed and chromatin was fragmented by partial digestion with Micrococcal Nuclease. ChIP was performed using normal rabbit IgG, or antibodies against H3K27Ac or BRD4. Purified DNA was subjected to quantitative PCR (qPCR) using the primer pairs listed in Supplementary Table S3. Protein enrichment was expressed as a percentage of the input. All experiments were performed in triplicate.

Luciferase reporter assay

The luciferase reporter assay was carried out as described previously (20). Luciferase reporter plasmids were constructed by inserting the 3′UTRs of BRD4, CREBBP, and FRAT2 downstream of the luciferase gene in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). All site-specific mutations were generated by the KOD -Plus- Mutagenesis Kit (TOYOBO).

RNA immunoprecipitation analysis

RNA immunoprecipitation (RIP) assay was carried out using the RiboCluster Profiler RIP assay kit for miRNA (MBL International) as described previously (24). Briefly, cells were lysed 24 hours after transfection of miR-NC or miR-766-5p, and incubated with AGO2 antibody-immobilized beads or normal rabbit IgG antibody-immobilized beads for RIP. RNA was isolated from each bead complex. For RIP-PCR analysis, cDNA was synthesized from RNA isolated by AGO2-RIP or IgG-RIP and from total RNA (input), and quantitative RT-PCR (qRT-PCR) was performed using primers listed in Supplementary Table S3.

Plasmid construction and transfection

The BRD4 expression vector was generated previously (20). Site-specific mutations were generated as described previously (20). The BRD4 expression vector was transfected into HCT116−/− cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. CBP cDNA were purchased from Kazusa DNA Research Institute (Chiba, Japan) and were subcloned into the pFN28A HaloTag CMV-neo Flexi Vector (Promega) to generate a mammalian expression vector.

In vivo xenograft model in HCT116−/− cells

In vivo miRNA administration was performed as described previously (20). Six-week-old female BALB/c nude mice were purchased from Oriental Bio Service, Japan. Briefly, a total of 2.0 × 106 cells in 100 μL of PBS were subcutaneously injected into the dorsal side of the mice. A mixture of 1 nmol of miR-NC or miR-766-5p and 100 μL of AteloGene (KOKEN) was administered around the tumor on days 3, 6, 10, and 13. The tumor volume was calculated using the following formula: (shortest diameter)2 × (longest diameter) × 0.5. All experimental protocols involving mice were approved by the Tokyo Medical and Dental University (TMDU) Animal Care and Use Committee.

In vivo xenograft model in MIAPaCa2 and Ty82-JQ1R cells

A total of 5.0 × 106 MIAPaCA2 cells or 2.0 × 106 Ty82-JQ1R cells in 100 μL of PBS were subcutaneously injected into the dorsal side of the 6-week-old female BALB/c nude mice. To formulate the ointment incorporating miRNAs, the ionic liquid transdermal system (ILTS; MEDRx) was used as described previously (24). ILTS increases the transdermal permeability of nucleotides, including miRNAs, in the hydrophobic field of skin tissue to achieve efficient delivery into subcutaneous tumor cells (24). The 0.2% ointment incorporating miR-NC or miR-766-5p was formulated (2 mg of miRNA/mL of ointment) and 10–20 μL of ointment (20–40 μg of miRNA) was locally applied daily onto subcutaneous tumors. All experimental protocols involving mice were approved by the TMDU Animal Care and Use Committee.

Immunohistochemistry

IHC was performed as described previously (20). Anti-NUT was used to detect BRD4-NUT expression. IHC staining was analyzed using an IHC image analysis toolbox for ImageJ software (NIH, Bethesda, MD). Briefly, the software was first trained to build a statistical color detection model based on the positive-staining pixels of each protein. The positive-staining area was calculated in each of three images of sections from three tumors treated with miR-NC or miR-766-5p. The results were normalized to the mean positive-staining area of tumors treated with miR-NC.

Public database analysis

To explore the expression of miR-766-5p, miR-34a-5p, let-7a-5p, BRD4, CREBBP, FRAT2, and MYC in various types of cancer, miRNA-sequencing and RNA-sequencing data of The Cancer Gene Atlas (TCGA) project were obtained from ENCORI database (http://starbase.sysu.edu.cn/index.php).

Statistical analysis

Differences between groups were assessed with the Student t test, one-way ANOVA with Bonferroni adjustment, or Mann–Whitney U test as appropriate. All statistical analyses were carried out with R software. P values < 0.05 were considered significant.

miR-766-5p was identified as a miRNA-suppressing MYC expression in cancer cells

First, we screened the effects of 138 tumor-suppressive miRNAs, which were extracted in the previous study (19), on the MYC reporter activity (Fig. 1A). As p53 suppressed the expression of MYC (Supplementary Fig. S1A and S1B), as reported previously (25), we used HCT116−/− cells for this screening to identify miRNAs suppressing MYC regardless of p53 status (Fig. 1A). As a result, 12 miRNAs suppressed MYC reporter activity to less than 0.4 compared with miR-NC (Fig. 1B; Supplementary Table S4). Among 12 miRNAs, miR-766-5p, miR-7160, and miR-3140–3p suppressed the expression of MYC to less than 0.5 compared with miR-NC (Fig. 1C). On Western blotting, the protein expression of MYC was most suppressed by miR-766-5p among these 3 miRNAs in HCT116−/− cells (Fig. 1D). Similar results were also observed in HCT116+/+ cells (Supplementary Fig. S2A and S2B).

Figure 1.

miR-766-5p was identified as a miRNA that suppressed MYC expression in cancer cells. A, The strategy used to identify tumor-suppressive miRNAs that suppressed MYC expression. B, Top, MYC reporter activity was evaluated in HCT116−/− cells using a relative ratio compared with that of miR-NC–transfected cells. Bottom, 12 miRNAs that suppressed MYC reporter activity to less than 0.4 compared with miR-NC. C, qRT-PCR of MYC mRNA in HCT116−/− cells transfected with indicated miRNAs. The expression of MYC was assessed using a relative ratio compared with that of miR-NC–transfected cells. Bar, SD for triplicate experiments. D, Western blot analysis of MYC in HCT116−/− cells after transfection with 10 nmol/L of miR-NC, miR-766-5p, miR-7160–5p, or miR-3140–3p. The intensity of MYC bands was quantified by densitometry using ImageJ software and is shown as the fold change after normalization with β-actin. E and F, Western blot analysis of MYC (E) and cell growth assay (F) in indicated cell lines after transfection with 10 nmol/L of miR-NC or miR-766-5p. The intensity of MYC bands was quantified by densitometry and is shown as the fold change after normalization with β-actin. The cell growth rate was assessed by the crystal violet staining assay using a relative ratio compared with day 0. Bar, SD for triplicate experiments. *, P < 0.05.

Figure 1.

miR-766-5p was identified as a miRNA that suppressed MYC expression in cancer cells. A, The strategy used to identify tumor-suppressive miRNAs that suppressed MYC expression. B, Top, MYC reporter activity was evaluated in HCT116−/− cells using a relative ratio compared with that of miR-NC–transfected cells. Bottom, 12 miRNAs that suppressed MYC reporter activity to less than 0.4 compared with miR-NC. C, qRT-PCR of MYC mRNA in HCT116−/− cells transfected with indicated miRNAs. The expression of MYC was assessed using a relative ratio compared with that of miR-NC–transfected cells. Bar, SD for triplicate experiments. D, Western blot analysis of MYC in HCT116−/− cells after transfection with 10 nmol/L of miR-NC, miR-766-5p, miR-7160–5p, or miR-3140–3p. The intensity of MYC bands was quantified by densitometry using ImageJ software and is shown as the fold change after normalization with β-actin. E and F, Western blot analysis of MYC (E) and cell growth assay (F) in indicated cell lines after transfection with 10 nmol/L of miR-NC or miR-766-5p. The intensity of MYC bands was quantified by densitometry and is shown as the fold change after normalization with β-actin. The cell growth rate was assessed by the crystal violet staining assay using a relative ratio compared with day 0. Bar, SD for triplicate experiments. *, P < 0.05.

Close modal

Next, we compared the effects of miR-766-5p on the expression of MYC in different types of cancer cells (HCT116+/+, HCT116−/−, MIAPaCa2, AGS, A549, KYSE150, and HSC2 cells) and noncancer cells (TIG1, HUVEC, RT7, and Het1A). As shown in Fig. 1E, miR-766-5p downregulated the expression of MYC in all cancer cells tested except for A549 cells, whereas miR-766-5p did not downregulate MYC expression in noncancer cells whose MYC expression was intrinsically low. Accordingly, miR-766-5p suppressed cancer cell growth in vitro, whereas the growth-suppressive effects of miR-766-5p on noncancer cells were limited (Fig. 1F; Supplementary Fig. S2C).

miR-766-5p directly targets BRD4 and CBP

To explore the target genes of miR-766-5p, we performed gene expression array analysis. As MYC expression was markedly suppressed by miR-766-5p in HCT116−/− and MIAPaCa2 cells (Fig. 1E), we selected these two cell lines for the expression analysis. Transfection of miR-766-5p upregulated 1,756 and 1,814 genes, and downregulated 1,313 and 1,746 genes in HCT116−/− and MIAPaCa2 cells, respectively (fold change > 1.5; Supplementary Fig. S3). Gene-set enrichment analysis (GSEA) of gene expression array data revealed that the signatures of MYC targets, glycolysis, and WNT/β-catenin signaling were commonly downregulated by miR-766-5p in both cell lines (Fig. 2A). The amount of l-lactate, the end product of glycolysis, was reduced by miR-766-5p or knockdown of MYC in HCT116−/− and MIAPaCa2 cells, suggesting that miR-766-5p downregulated glycolysis via MYC suppression (Fig. 2B and C).

Figure 2.

Gene expression analysis in HCT116−/− and MIAPaCa2 cells transfected with miR-766-5p. A, GSEA of gene expression array data in HCT116−/− and MIAPaCa2 cells transfected with 10 nmol/L of miR-NC- and miR-766-5p. GSEA plot showing the enrichment of gene signatures associated with MYC targets, glycolysis, and WNT/β-catenin signaling between miR-NC– and miR-766-5p–transfected cells. FDR, false discovery rate. B and C, Relative levels of l-lactate in HCT116−/− and MIAPaCa2 cells transfected with 10 nmol/L of miR-NC or miR-766-5p (B), or 20 nmol/L of si-NC or si-MYC (C). Bar, SD for triplicate experiments. *, P < 0.05. D, Top left, the Venn diagram shows that 515 genes were commonly downregulated (fold change >1.5) by transfection of miR-766-5p in HCT116−/− and MIAPaCa2 cells. Right, the Venn diagram shows that 212 genes were predicted as candidate 3′UTR-targets of miR-766-5p by the TargetScan program. Bottom, the Venn diagram shows that 183 genes were predicted as candidate coding sequence (CDS)-targets of miR-766-5p by the StarMirDB program. In total, 324 candidate genes (3′UTR, 141 genes; CDS, 112 genes; both 3′UTR and CDS, 71 genes) were identified as targets of miR-766-5p (Supplementary Table S1).

Figure 2.

Gene expression analysis in HCT116−/− and MIAPaCa2 cells transfected with miR-766-5p. A, GSEA of gene expression array data in HCT116−/− and MIAPaCa2 cells transfected with 10 nmol/L of miR-NC- and miR-766-5p. GSEA plot showing the enrichment of gene signatures associated with MYC targets, glycolysis, and WNT/β-catenin signaling between miR-NC– and miR-766-5p–transfected cells. FDR, false discovery rate. B and C, Relative levels of l-lactate in HCT116−/− and MIAPaCa2 cells transfected with 10 nmol/L of miR-NC or miR-766-5p (B), or 20 nmol/L of si-NC or si-MYC (C). Bar, SD for triplicate experiments. *, P < 0.05. D, Top left, the Venn diagram shows that 515 genes were commonly downregulated (fold change >1.5) by transfection of miR-766-5p in HCT116−/− and MIAPaCa2 cells. Right, the Venn diagram shows that 212 genes were predicted as candidate 3′UTR-targets of miR-766-5p by the TargetScan program. Bottom, the Venn diagram shows that 183 genes were predicted as candidate coding sequence (CDS)-targets of miR-766-5p by the StarMirDB program. In total, 324 candidate genes (3′UTR, 141 genes; CDS, 112 genes; both 3′UTR and CDS, 71 genes) were identified as targets of miR-766-5p (Supplementary Table S1).

Close modal

Among 515 commonly downregulated genes by miR-766-5p in HCT116−/− and MIAPaCa2 cells, 324 were predicted to be direct targets of miR-766-5p via the 3′UTR (141 genes), CDS (112 genes), or 3′UTR and CDS (71 genes) according to the TargetScan program (http://www.targetscan.org) and STarMirDB database (http://sfold.wadsworth.org; Fig. 2D; Supplementary Table S5; ref. 26). Unexpectedly, MYC was not included in 324 candidate genes due to lack of target sequences of miR-766-5p in 3′UTR and CDS. As only BRD4 and CREBBP were reported to promote MYC transcription directly among 324 candidate target genes of miR-766-5p (Supplementary Table S5; refs. 9, 10, 13, 27–35), we focused on BRD4 and CREBBP. BRD4 and CBP are associated with SEs to drive transcription of MYC in cancer cells. As shown in Fig. 3A and Supplementary Fig. S4, BRD4 and CBP were reduced by miR-766-5p in all cell lines tested.

Figure 3.

miR-766-5p directly targeted CBP and BRD4. A, Western blot analysis of CBP and BRD4 in HCT116−/− and MIAPaCa2 cells 48 hours after transfection with 10 nmol/L of miR-NC, or miR-766-5p. The numbers under the blots correspond to densitometric analysis of each protein normalized to β-actin. The results are expressed as fold change relative to the miR-NC control. B, Luciferase reporter assays. HCT116–Lu cells were cotransfected with pmirGLO dual-luciferase vectors containing the wild-type (WT) 3′UTRs of CREBBP or mutant variants (Mt) of CREBBP and miR-NC or miR-766-5p. Top, a putative binding site of miR-766-5p within the 3′UTR of CREBBP and mutant sequences. Bottom, the results of the luciferase assay. C, Western blot analysis of BRD4 in HCT116−/− cells. Cells were cotransfected with the WT or MT BRD4 expression vector, and after 24 hours, 10 nmol/L of either miR-NC or miR-766-5p was additionally transfected. Top, putative binding sites of miR-766-5p within the coding sequence (CDS; R1 ∼ R5) and 3′UTR (R6) of BRD4. R1, R2, and R5 (black arrow), but not R3, R4, or R6 (white arrow), were considered to be directly targeted by miR-766-5p (see also Supplementary Fig S5 and S6). Bottom, Western blotting. D and E, Western blot analysis of MYC (D) and qRT-PCR of MYC mRNA (E) in HCT116−/−, MIAPaCa2, TIG1, and HUVEC cells 48 hours after transfection with siRNA [si-NC (40 nmol/L); si-BRD4, si-CREBBP, si-BRD4, and si-CREBBP (each 20 nmol/L)]. The intensity of MYC bands was quantified by densitometry using ImageJ software and is shown as the fold change after normalization with β-actin (D). na, not applicable. The expression of MYC was assessed using a relative ratio compared with that of si-NC–transfected cells (E). Bar, SD for triplicate experiments. F, Relative cell growth of HCT116−/−, MIAPaCa2, TIG1, and HUVEC cells transfected with indicated siRNAs. The cell growth rate was assessed by the CV staining assay using a relative ratio compared with that of si-NC–transfected cells. Bar, SD for triplicate experiments. NS, not significant. *, P < 0.05.

Figure 3.

miR-766-5p directly targeted CBP and BRD4. A, Western blot analysis of CBP and BRD4 in HCT116−/− and MIAPaCa2 cells 48 hours after transfection with 10 nmol/L of miR-NC, or miR-766-5p. The numbers under the blots correspond to densitometric analysis of each protein normalized to β-actin. The results are expressed as fold change relative to the miR-NC control. B, Luciferase reporter assays. HCT116–Lu cells were cotransfected with pmirGLO dual-luciferase vectors containing the wild-type (WT) 3′UTRs of CREBBP or mutant variants (Mt) of CREBBP and miR-NC or miR-766-5p. Top, a putative binding site of miR-766-5p within the 3′UTR of CREBBP and mutant sequences. Bottom, the results of the luciferase assay. C, Western blot analysis of BRD4 in HCT116−/− cells. Cells were cotransfected with the WT or MT BRD4 expression vector, and after 24 hours, 10 nmol/L of either miR-NC or miR-766-5p was additionally transfected. Top, putative binding sites of miR-766-5p within the coding sequence (CDS; R1 ∼ R5) and 3′UTR (R6) of BRD4. R1, R2, and R5 (black arrow), but not R3, R4, or R6 (white arrow), were considered to be directly targeted by miR-766-5p (see also Supplementary Fig S5 and S6). Bottom, Western blotting. D and E, Western blot analysis of MYC (D) and qRT-PCR of MYC mRNA (E) in HCT116−/−, MIAPaCa2, TIG1, and HUVEC cells 48 hours after transfection with siRNA [si-NC (40 nmol/L); si-BRD4, si-CREBBP, si-BRD4, and si-CREBBP (each 20 nmol/L)]. The intensity of MYC bands was quantified by densitometry using ImageJ software and is shown as the fold change after normalization with β-actin (D). na, not applicable. The expression of MYC was assessed using a relative ratio compared with that of si-NC–transfected cells (E). Bar, SD for triplicate experiments. F, Relative cell growth of HCT116−/−, MIAPaCa2, TIG1, and HUVEC cells transfected with indicated siRNAs. The cell growth rate was assessed by the CV staining assay using a relative ratio compared with that of si-NC–transfected cells. Bar, SD for triplicate experiments. NS, not significant. *, P < 0.05.

Close modal

In luciferase assays using reporter plasmid vectors containing wild-type (WT) or mutant (Mt) seed sequences of the 3′ UTR of CREBBP, the luciferase activity of the WT vector of CREBBP 3′UTR was reduced compared with that of the empty vector (EV) in miR-766-5p–transfected cells and was restored with the Mt vector, demonstrating that miR-766-5p directly targeted the 3′UTR of CREBBP (Fig. 3B). Similarly, the luciferase activity of the WT vectors of BRD4 CDS R1, R2, and R5, but not those of CDS R3, R4, and 3′UTR (R6), were reduced, whereas it was restored with the Mt vector (Supplementary Fig. S5A–S5F). These results suggested that miR-766-5p potentially targeted BRD4 CDS R1, R2, and R5. To confirm whether miR-766-5p targeted these CDS regions of BRD4, the WT Halotag-BRD4 expression vector or its synonymous Mt vectors with each region (R1, R2, R5, or combination of R1, R2, and R5 (R1/R2/R5)) were transfected into HCT116−/− cells, followed by transfection with miR-NC or miR-766-5p (Supplementary Fig. S6). As shown in Fig. 3C, the expression level of exogenously expressed BRD4-WT decreased in the miR-766-5p–transfected cells, whereas this miR-766-5p–induced reduction of exogenous BRD4 was restored by the mutations at R1, R2, R5, or R1/R2/R5. This suggested that miR-766-5p targeted these CDS regions of BRD4. Moreover, RIP-PCR analysis showed that BRD4 and CREBBP mRNA was enriched by RIP with AGO2 antibody after transfection of miR-766-5p, whereas MYC mRNA was not (Supplementary Fig. S7A and S7B). Collectively, miR-766-5p directly targeted BRD4 and CREBBP.

Concurrent suppression of BRD4 and CBP downregulated MYC expression in MYC-overexpressing cancer cells

We next examined the effects of suppression of BRD4 and CBP using siRNA targeting of each gene (si-BRD4 and si-CREBBP) on the expression of MYC. As shown in Fig. 3D and Supplementary Fig. S8A, si-BRD4 in combination with si-CREBBP led to more marked suppression of MYC expression in MIAPaCA2, KYSE150, and HSC2 cells, although these synergistic effects of the combination of si-BRD4 and si-CREBBP were not significant in HCT116+/+, HCT116−/−, and AGS cells because of the strong suppression of MYC expression by si-CREBBP. Note that the effects of combined knockdown of BRD4 and CBP on MYC suppression were limited in noncancer cells and A549 cells that had intrinsically low MYC expression (Fig. 3D; Supplementary Fig. S8A). Collectively, the concurrent suppression of BRD4 and CBP efficiently downregulated MYC expression in MYC-overexpressing cancer cells. In addition, the effects of the combined suppression of BRD4 with CBP on the cell growth were more notable in MYC-overexpressing cancer cells than in noncancer cells (Fig. 3F; Supplementary Fig. S8B). This suggested that concurrent downregulation of BRD4 and CBP is one of the tumor-suppressive mechanisms of miR-766-5p. Moreover, BRD4 or CBP overexpression partially rescued miR-766-5p overexpression–induced reduction of MYC expression in HCT116−/− cells (Supplementary Fig. S9).

miR-766-5p suppressed SEs via the downregulation of CBP in HCT116−/− cells

We next examined the effects of CBP suppression on the activity of SEs using H3K27Ac ChIP assay in HCT116−/− cells. SEs were analyzed previously in detail in HCT116 cells (Fig. 4A; refs. 1, 5). As shown in Fig. 4B, ChIP-qPCR analysis using H3K27Ac antibody revealed high levels of H3K27Ac at MYC-SEs in HCT116−/− cells. The knockdown of CBP significantly reduced H3K27Ac at MYC-SEs, suggesting that si-CREBBP reduced the activity of MYC-SEs. Furthermore, BRD4 ChIP assay demonstrated that the recruitment of BRD4 at MYC-SEs was reduced after knocking down CBP. Concordantly, lower levels of H3K27Ac at MYC-SEs were observed in miR-766-5p–transfected cells (Fig. 4C). This suggested that miR-766-5p reduced the activity of MYC-SEs via CBP suppression.

Figure 4.

miR-766-5p suppressed the activity of SEs in HCT116−/− cells. A, Location of the PCR amplicons at the MYC locus used for the ChIP-qPCR. qPCR was performed using input and ChIP DNAs. Red lines indicate MYC super-enhancer regions according to a previous report (1). B and C, H3K27Ac ChIP-qPCR or BRD4 ChIP-qPCR at the indicated loci in HCT116−/− cells transfected with si-NC or si-CREBBP (B), and H3K27Ac ChIP-qPCR at the indicated loci in HCT116−/− cells transfected with miR-NC or miR-766-5p (C). Relative ChIP enrichment values in the indicated regions are expressed as percentages relative to input DNA. Bar, SD for triplicate experiments. D, qRT-PCR of indicated genes in HCT116−/− cells transfected with 10 nmol/L of miR-NC or miR-766-5p. The expression of each gene was assessed using a relative ratio compared with that of miR-NC–transfected cells. Bar, SD for triplicate experiments. E, H3K27Ac ChIP-qPCR at the IRS1, TNS4, and UCA1 loci in HCT116−/− cells transfected with miR-NC or miR-766-5p. Left, location of the PCR amplicons in each gene. Red lines indicate SE regions. Right, relative ChIP enrichment values in the indicated regions are expressed as percentages relative to input DNA. Bar, SD for triplicate experiments. *, P < 0.05.

Figure 4.

miR-766-5p suppressed the activity of SEs in HCT116−/− cells. A, Location of the PCR amplicons at the MYC locus used for the ChIP-qPCR. qPCR was performed using input and ChIP DNAs. Red lines indicate MYC super-enhancer regions according to a previous report (1). B and C, H3K27Ac ChIP-qPCR or BRD4 ChIP-qPCR at the indicated loci in HCT116−/− cells transfected with si-NC or si-CREBBP (B), and H3K27Ac ChIP-qPCR at the indicated loci in HCT116−/− cells transfected with miR-NC or miR-766-5p (C). Relative ChIP enrichment values in the indicated regions are expressed as percentages relative to input DNA. Bar, SD for triplicate experiments. D, qRT-PCR of indicated genes in HCT116−/− cells transfected with 10 nmol/L of miR-NC or miR-766-5p. The expression of each gene was assessed using a relative ratio compared with that of miR-NC–transfected cells. Bar, SD for triplicate experiments. E, H3K27Ac ChIP-qPCR at the IRS1, TNS4, and UCA1 loci in HCT116−/− cells transfected with miR-NC or miR-766-5p. Left, location of the PCR amplicons in each gene. Red lines indicate SE regions. Right, relative ChIP enrichment values in the indicated regions are expressed as percentages relative to input DNA. Bar, SD for triplicate experiments. *, P < 0.05.

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According to a previous report (1), several tumor-associated genes, including IRS1, TNS4, UCA1, ACLS5, FAM83H, and PAQR4, which were not candidate targets of miR-766-5p (Supplementary Table S5), were also SE-marked genes in HCT116 cells. As shown in Fig. 4D, the mRNA expression of these genes was significantly reduced by the overexpression of miR-766-5p. Furthermore, H3K27Ac ChIP assay demonstrated lower levels of H3K27Ac at IRS1-, TNS4-, and UCA1-SEs after miR-766-5p transfection (Fig. 4E). Taken together, miR-766-5p reduced the activity of SEs in HCT116−/− cells.

miR-766-5p suppressed WNT/β-catenin signaling by targeting FRAT2 or CBP

Next, we examined the effects of miR-766-5p on WNT/β-catenin signaling using the TCF/LEF reporter assay. As shown in Fig. 5A, TCF/LEF reporter activity was reduced by miR-766-5p in HCT116−/− and MIAPaCa2 cells, suggesting that miR-766-5p inhibited WNT/β-catenin signaling. Similar results were also observed in AGS, SNU398, and MKN28 cells, which exhibited high WNT/β-catenin signaling activity due to CTNNB1 or APC mutation (https://portals.broadinstitute.org/ccle; Supplementary Fig. S10A). CREBBP, FRAT2, FZD7, DVL2, SMAD4, and CSNK2A1 genes, which were predicted target genes of miR-766-5p, were associated with WNT/β-catenin signaling, although the expression of FZD7 and CSNK2A1 was not suppressed by miR-766-5p in HCT116−/− cells (Supplementary Fig. S10B and S10C). Among these genes, si-CREBBP and si-FRAT2 reduced the activity of the TCF/LEF reporter in HCT116−/−, MIAPaCa2, AGS, SNU398, and MKN28 cells (Fig. 5B; Supplementray Fig. S10D). On Western blotting, the expression of FRAT2 was reduced in miR-766-5p–transfected HCT116−/−, MIAPaCa2, SNU398, and MKN28 cells, but not in AGS cells with intrinsically low FRAT2 expression (Fig. 5C; Supplementray Fig. S10E). The luciferase reporter activity of the WT vector of FRAT2 3′UTR was lower than that of the empty vector (EV) in miR-766-5p–transfected cells and was restored by the MT vector (Fig. 5D). This suggested that miR-766-5p directly targeted the 3′UTR of FRAT2. Taken together, miR-766-5p suppressed WNT/β-catenin signaling, at least in part, via CREBBP or FRAT2.

Figure 5.

miR-766-5p suppressed WNT/β-catenin signaling via the downregulation of CBP or FRAT2. A and B, TCF/LEF reporter assay in HCT116−/− and MIAPaCa2 transfected with 10 nmol/L of miR-NC or miR-766-5p (A), and 20 nmol/L of si-NC or indicated siRNAs (B). Relative TCF/LEF activity was assessed using a relative ratio compared with that of miR-NC- or si-NC–transfected cells. Bar, SD for triplicate experiments. C, Western blot analysis of FRAT2 in HCT116−/− and MIAPaCa2 cells 48 hours after transfection with 10 nmol/L of miR-NC, or miR-766-5p. The numbers under the blots correspond to densitometric analysis of each protein normalized to β-actin. The results are expressed as fold change relative to the miR-NC control. D, Luciferase reporter assays. HCT116−/− cells were cotransfected with pmirGLO dual-luciferase vectors containing the wild-type (WT) 3′UTRs of FRAT2 or mutant variants (Mt) of FRAT2 and miR-NC or miR-766-5p. Top, a putative binding site of miR-766-5p within the 3′UTR of FRAT2 and mutant sequences. Bottom, the results of the luciferase assay. E, Western blot analysis (top) and cell growth assay (bottom) of HCT116−/− and MIAPaCa2 cells after treatment with DMSO, JQ1, or the combination of JQ1 and PKF118–310 (β-catenin inhibitor). The cell growth rate was assessed by crystal violet staining using a relative ratio compared with that of DMSO-treated cells. Bar, SD for triplicate experiments. NS, not significant. *, P < 0.05.

Figure 5.

miR-766-5p suppressed WNT/β-catenin signaling via the downregulation of CBP or FRAT2. A and B, TCF/LEF reporter assay in HCT116−/− and MIAPaCa2 transfected with 10 nmol/L of miR-NC or miR-766-5p (A), and 20 nmol/L of si-NC or indicated siRNAs (B). Relative TCF/LEF activity was assessed using a relative ratio compared with that of miR-NC- or si-NC–transfected cells. Bar, SD for triplicate experiments. C, Western blot analysis of FRAT2 in HCT116−/− and MIAPaCa2 cells 48 hours after transfection with 10 nmol/L of miR-NC, or miR-766-5p. The numbers under the blots correspond to densitometric analysis of each protein normalized to β-actin. The results are expressed as fold change relative to the miR-NC control. D, Luciferase reporter assays. HCT116−/− cells were cotransfected with pmirGLO dual-luciferase vectors containing the wild-type (WT) 3′UTRs of FRAT2 or mutant variants (Mt) of FRAT2 and miR-NC or miR-766-5p. Top, a putative binding site of miR-766-5p within the 3′UTR of FRAT2 and mutant sequences. Bottom, the results of the luciferase assay. E, Western blot analysis (top) and cell growth assay (bottom) of HCT116−/− and MIAPaCa2 cells after treatment with DMSO, JQ1, or the combination of JQ1 and PKF118–310 (β-catenin inhibitor). The cell growth rate was assessed by crystal violet staining using a relative ratio compared with that of DMSO-treated cells. Bar, SD for triplicate experiments. NS, not significant. *, P < 0.05.

Close modal

As miR-766-5p inhibited both SE and WNT/β-catenin signaling activity, we examined the effects of cotargeting SEs and WNT/β-catenin signaling on the expression of MYC using the BET inhibitor JQ1 and β-catenin inhibitor PKF118–310. As shown in Fig. 5E and Supplementray Fig. S10F, the combination of JQ1 and PKF118–310 cooperatively downregulated the expression of MYC, and suppressed the in vitro tumor growth in HCT116−/−, MIAPaCa2, and AGS cells.

Local administration of miR-766-5p suppressed in vivo tumor growth

In vivo therapeutic effects of miR-766-5p were evaluated using a xenograft mouse model of HCT116−/− cells. miR-NC or miR-766-5p was administered into the subcutaneous space around tumors 4 times (3, 6, 10, and 13 days after the injection of cells (Fig. 6A). As shown in Fig. 6BD, tumors treated with miR-766-5p at 15 days after the inoculation of HCT116−/− cells were significantly smaller than those treated with miR-NC. No notable adverse events, including body weight loss or skin damage, were observed around the administration site of miRNAs (Fig. 6E). On qRT-PCR, the expression of miR-766-5p was significantly higher in the miR-766-5p–treated tumors than in the miR-NC–treated tumors, suggesting that miR-766-5p was delivered into the miR-766-5p–treated tumors (Fig. 6F). IHC staining demonstrated that the expression levels of BRD4, CBP, and MYC decreased in the resected tumors treated with miR-766-5p (Fig. 6G; Supplementray Fig. S11). Similarly, administration of miR-766-5p using ILTS delivery inhibited tumor growth in nude mice that were subcutaneously inoculated with MIAPaCa2 cells (Supplementary Fig. S12A–S12H). This suggested that miR-766-5p suppressed in vivo tumor cell growth by downregulating BRD4 and CBP.

Figure 6.

In vivo therapeutic effects of miR-766-5p in HCT116−/− cells. A, The in vivo experimental schedule. Tumors were formed by subcutaneous injection of HCT116−/− cells into nude mice. miR-NC or miR-766-5p was administered subcutaneously around the tumors for a total of four times (3, 6, 10, and 13 days after the injection of cells). B, Representative images of tumor-bearing nude mice and resected tumors at 15 days after the injection of HCT116−/− cells. Scale bar, 10 mm. C, Tumor growth curves of xenograft mouse models treated with miR-NC or miR-766-5p (n = 4 each). The tumor volume was calculated using the following formula: (shortest diameter) 2 × (longest diameter) × 0.5. Bar, SD for 4 mice; *, P < 0.05. D, Weights of the resected tumors. Tumor weights are shown as a box plot. Bar, SD for 4 mice; *, P < 0.05. E, Body weights on day 3 (before the treatment) and on day 15 (after the treatment) were measured. F, qRT-PCR of miR-766-5p in the resected tumors. Each experiment was performed in triplicate. The relative ratio was normalized to the expression of RNU6B. Bar, SD for 4 mice; *, P < 0.05. G, Representative images of immunohistochemical staining for BRD4, CBP, and MYC in resected tumors. Original magnification, ×200.

Figure 6.

In vivo therapeutic effects of miR-766-5p in HCT116−/− cells. A, The in vivo experimental schedule. Tumors were formed by subcutaneous injection of HCT116−/− cells into nude mice. miR-NC or miR-766-5p was administered subcutaneously around the tumors for a total of four times (3, 6, 10, and 13 days after the injection of cells). B, Representative images of tumor-bearing nude mice and resected tumors at 15 days after the injection of HCT116−/− cells. Scale bar, 10 mm. C, Tumor growth curves of xenograft mouse models treated with miR-NC or miR-766-5p (n = 4 each). The tumor volume was calculated using the following formula: (shortest diameter) 2 × (longest diameter) × 0.5. Bar, SD for 4 mice; *, P < 0.05. D, Weights of the resected tumors. Tumor weights are shown as a box plot. Bar, SD for 4 mice; *, P < 0.05. E, Body weights on day 3 (before the treatment) and on day 15 (after the treatment) were measured. F, qRT-PCR of miR-766-5p in the resected tumors. Each experiment was performed in triplicate. The relative ratio was normalized to the expression of RNU6B. Bar, SD for 4 mice; *, P < 0.05. G, Representative images of immunohistochemical staining for BRD4, CBP, and MYC in resected tumors. Original magnification, ×200.

Close modal

miR-766-5p suppressed tumor growth of NUT midline carcinoma

Considering that miR-766-5p suppressed BRD4 via CDS, we examined whether miR-766-5p downregulated the BRD4-NUT fusion protein that drives NUT midline carcinoma (NMC) via its transcriptionally active mega domain with high levels of H3K27Ac (36). We used Ty82-JQ1R cells, which were generated from Ty82 cells harboring BRD4-NUT due to t(15,19) (refs. 20, 37), as these cells are transplantable into nude mice. As shown in Fig. 7A, miR-766-5p suppressed in vitro tumor growth with downregulation of the expression of BRD4-NUT, CBP, and MYC in Ty82-JQ1R cells. Knockdown experiments revealed that suppression of BRD4-NUT and CBP cooperatively inhibited in vitro tumor cell growth in Ty82-JQ1R cells (Fig. 7B). WNT/β-catenin signaling was not affected by miR-766-5p in Ty82-JQ1R cells, as the activity of TCF/LEF reporter was hardly detected in these cells. In the xenograft mouse model, local administration of miR-766-5p using ILTS delivery significantly suppressed in vivo tumor growth of Ty82-JQ1R cells with no notable adverse events (Fig. 7CG). On the basis of qRT-PCR and IHC, miR-766-5p was highly expressed with reduced expression of BRD4-NUT, CBP, and MYC in miR-766-5p–treated tumors (Fig. 7H and I; Supplementary Fig. S13). Collectively, miR-766-5p suppressed the in vivo tumor growth of Ty82-JQ1R cells by downregulating BRD4-NUT and CBP.

Figure 7.

In vitro and in vivo effects of miR-766-5p in NMC cells. A, Western blot analysis of indicated proteins (left) and cell growth assay (right), and in Ty82-JQ1R cells after transfection with 10 nmol/L of miR-NC or miR-766-5p. The numbers under the blots correspond to densitometric analysis of each protein normalized to β-actin. The results are expressed as fold change relative to the miR-NC control. The cell growth rate was assessed by crystal violet staining assay using a relative ratio compared with day 0. Bar, SD for triplicate experiments. *, P < 0.05. B, Western blot analysis of BRD4-NUT, CBP, and MYC expression (left), and cell growth assay (right) in Ty82-JQ1R cells transfected with 10 nmol/L of si-NC, si-BRD4, si-CREBBP, or si-BRD4 and si-CREBBP. The intensity of MYC bands was quantified by densitometry and is shown as the fold change after normalization with β-actin. The cell growth rate was assessed 72 hours after transfection using a relative ratio compared with that of si-NC–transfected cells. Bar, SD for triplicate experiments. *, P < 0.05. C, The experimental schedule for miR-766-5p treatment using the ILTS in nude mice. On day 7 after inoculation of Ty82-JQ1R cells, the local administration of miR-NC or miR-766-5p to subcutaneous tumors was initiated. D, Representative images of tumor-bearing nude mice and resected tumors at 21 days after the injection of Ty82-JQ1R cells. Scale bar, 10 mm. E, Tumor growth curves of xenograft mouse models treated with miR-NC or miR-766-5p (n = 5, each). The tumor volume was calculated using the following formula: (shortest diameter) 2 × (longest diameter) × 0.5. Bar, SD for 5 mice; *, P < 0.05. F, Body weights on day 5 (before the treatment) and on day 21 (after the treatment) were measured. G, Weights of the resected tumors. Tumor weights are shown as a box plot. Bar, SD for 5 mice; *, P < 0.05. H, qRT-PCR of miR-766-5p in the resected tumors. Each experiment was performed in triplicate. The relative ratio was normalized to the expression of RNU6B. Bar, SD for 5 mice; *, P < 0.05. I, Representative images of IHC staining for BRD4-NUT, CBP, and MYC in resected tumors. Original magnification, ×200.

Figure 7.

In vitro and in vivo effects of miR-766-5p in NMC cells. A, Western blot analysis of indicated proteins (left) and cell growth assay (right), and in Ty82-JQ1R cells after transfection with 10 nmol/L of miR-NC or miR-766-5p. The numbers under the blots correspond to densitometric analysis of each protein normalized to β-actin. The results are expressed as fold change relative to the miR-NC control. The cell growth rate was assessed by crystal violet staining assay using a relative ratio compared with day 0. Bar, SD for triplicate experiments. *, P < 0.05. B, Western blot analysis of BRD4-NUT, CBP, and MYC expression (left), and cell growth assay (right) in Ty82-JQ1R cells transfected with 10 nmol/L of si-NC, si-BRD4, si-CREBBP, or si-BRD4 and si-CREBBP. The intensity of MYC bands was quantified by densitometry and is shown as the fold change after normalization with β-actin. The cell growth rate was assessed 72 hours after transfection using a relative ratio compared with that of si-NC–transfected cells. Bar, SD for triplicate experiments. *, P < 0.05. C, The experimental schedule for miR-766-5p treatment using the ILTS in nude mice. On day 7 after inoculation of Ty82-JQ1R cells, the local administration of miR-NC or miR-766-5p to subcutaneous tumors was initiated. D, Representative images of tumor-bearing nude mice and resected tumors at 21 days after the injection of Ty82-JQ1R cells. Scale bar, 10 mm. E, Tumor growth curves of xenograft mouse models treated with miR-NC or miR-766-5p (n = 5, each). The tumor volume was calculated using the following formula: (shortest diameter) 2 × (longest diameter) × 0.5. Bar, SD for 5 mice; *, P < 0.05. F, Body weights on day 5 (before the treatment) and on day 21 (after the treatment) were measured. G, Weights of the resected tumors. Tumor weights are shown as a box plot. Bar, SD for 5 mice; *, P < 0.05. H, qRT-PCR of miR-766-5p in the resected tumors. Each experiment was performed in triplicate. The relative ratio was normalized to the expression of RNU6B. Bar, SD for 5 mice; *, P < 0.05. I, Representative images of IHC staining for BRD4-NUT, CBP, and MYC in resected tumors. Original magnification, ×200.

Close modal

We identified miR-766-5p as a miRNA that downregulates MYC expression by inhibiting the activity of SEs via targeting CBP and BRD4 in cancer cells (Supplementary Fig. S14). Moreover, miR-766-5p suppressed WNT/β-catenin signaling partially by suppressing FRAT2 or CBP. In in vivo therapeutic models, the administration of miR-766-5p suppressed tumor cell growth in HCT116−/− and Ty82-JQ1R cells.

MYC is one of the most frequently overexpressed transcriptional factors in many types of cancer (7). It regulates the global transcription program that leads to the tumorigenic cellular phenotype (38). Several lines of experimental evidence support that the inactivation of MYC can repress tumor growth with tolerable toxicity in genetic mouse models, indicating MYC to be a potent therapeutic target for cancer (39, 40). However, MYC remains “undruggable” because of the lack of effective binding domains on its surface (41). Nucleic acid therapeutics using miRNAs that suppress MYC may be an alternative strategy for targeting MYC.

We revealed that miR-766-5p downregulated the levels of H3K27Ac at the MYC-SEs by targeting CBP. H3K27Ac is required for enhancer activation (42) and BRD4 binds to active enhancers to drive transcription. As miR-766-5p concurrently targets CBP and BRD4, miR-766-5p may efficiently suppress the expression of SE-marked genes other than MYC, such as IRS1, TNS4, and UCA1, in HCT116−/− cells. IRS1, a signaling adaptor protein, is associated with the activation of proliferative pathways, including PI3K and MAPK signaling, in cancer (43). TNS4 promotes tumor growth through β-catenin signaling in colon cancer (44). UCA1, a long noncoding RNA, was reported to promote tumorigenesis by sponging tumor-suppressive miRNAs in several cancers (45, 46). According to previous reports, a large number of the SE-marked genes have functions that are associated with cancer hallmarks (1). As these SE-marked genes are cancer cell type–specific and many types of cancer cells acquire cancer cell–specific SEs, targeting SEs by miR-766-5p may be rational for cancer therapeutics. To further understand the tumor-suppressive effects of miR-766-5p via epigenetic regulation, H3K27Ac ChIP-seq is required in several cancer and noncancer cells transfected with miR-NC or miR-766-5p in future study.

Moreover, we demonstrated that miR-766-5p suppresses WNT/β-catenin signaling, at least in part, via the suppression of CBP or FRAT2. Consistent with our results, CBP was reported to act as a coactivator interacting with β-catenin to promote WNT/β-catenin signaling (47–49). FRAT2 was reported to promote the accumulation of nuclear β-catenin via the inhibition of phosphorylation of β-catenin (30). Concordantly, knockdown of FRAT2 or transfection of miR-766-5p increased phosphorylated β-catenin and consequently inhibited the accumulation of nuclear β-catenin in HCT116−/− cells (Supplementary Fig. S15A–S15D). Furthermore, our preliminary study revealed that knockdown of BRD4 or JQ1 treatment upregulated TCF/LEF reporter activity in WNT/β-catenin–active cancer cells (Supplementary Fig. S16A and S16D). This upregulation of TCF/LEF activity was cancelled by combined treatment of JQ1 with PKF118–310, reducing the expression of MYC. Thus, the effect of JQ1 might be attenuated by increased Wnt/β-catenin signaling. Cotargeting of BRD4 and WNT/β-catenin signaling by miR-766-5p may be effective for inhibiting MYC in WNT/β-catenin–active cancer.

A recent study reported that MYC protein stability was negatively regulated by BRD4 (50). Although MYC mRNA expression was strongly suppressed by miR-766-5p via downregulation of CBP and BRD4, MYC protein stability might be upregulated by miR-766-5p via BRD4 suppression. Further analysis is required to reveal the effects of miR-766-5p on MYC protein stability.

In this study, we used TargetScan program and STarMirDB database for the prediction of 3′UTR-targets and CDS-targets of miR-766-5p, respectively. On the other hands, 17,100 genes were predicted to be targets of miR-766-5p via 3′UTR or CDS in miRWalk algorithm. Among 9792 genes predicted to be targets of miR-766-5p in TargetScan and STarMirDB, 8538 genes were included in the prediction of miRWalk program (Supplementary Fig S17A). Moreover, among 324 candidate targets of miR-766-5p in this study (Fig. 2D; Supplementary Table S5), 293 genes including BRD4, CREBBP and FRAT2 were overlapped in the prediction of miR-766-5p targets in miRWalk database (Supplementary Fig. S17B). This also supports the validity of our results.

According to the miRNA-seq data of TCGA study, the expression of miR-766-5p was extremely low in both various types of cancers and normal tissues compared with that of the representative miRNA miR-34a-5p or let-7a-5p (Supplementary Table S6). Correlation was not observed between the expression level of miR-766-5p and that of BRD4, CREBBP, FRAT2 or MYC in colon adenocarcinoma of TCGA data (Supplementary Fig. S18). In addition, the expression of miR-766-5p was extremely low in a panel of cell lines and normal tissues, whereas that of miR-766-5p increased thousands of times in the miR-766-5p–transfected cells (Supplementary Fig. S19A–S19C). These results suggested that the expression of endogenous miR-766-5p might be too low to function in clinical samples. According to TCGA data, the expression of miR-766-5p was not correlated with overall survival in colon, pancreatic, stomach, esophageal, head and neck, and lung cancers (Supplementary Fig. S20).

Finally, we confirmed that miR-766-5p suppressed tumor growth of Ty82-JQ1R NMC cells via the suppression of BRD4-NUT and CBP in vitro and in vivo. Recent clinical trials reported that several BET inhibitors, including OTX015 and RO6870810, exhibited therapeutic potential in NMC among solid tumors (51–53). In addition, the combination of a BET inhibitor with CBP/p300 inhibitors exerted synergistic antitumor effects in NMC (54, 55). Concordantly, we demonstrated that the downregulation of BRD4-NUT and CBP cooperatively suppressed in vitro tumor cell growth in Ty82-JQ1R cells. Administration of miR-766-5p may be efficient for inhibiting tumor cell growth in MYC-driven tumors such as NMC. For the development of miR-766-5p-based therapeutics, effective drug delivery systems and chemical modification of miRNAs are required. In conclusion, miR-766-5p suppressed the expression of MYC by targeting CBP and BRD4. Targeting SEs using miR-766-5p–based therapeutics may be reasonable for the treatment of MYC-driven cancer.

Y. Gen reports grants from Ministry of Education, Culture, Sports, Science and Technology (MEXT) during the conduct of the study. J. Inazawa reports grants from Ministry of Education, Culture, Sports, Science and Technology (MEXT) and grants from Japan Agency for Medical Research and Development (AMED) during the conduct of the study. No disclosures were reported by the other authors.

Y. Gen: Conceptualization, formal analysis, funding acquisition, investigation, methodology, writing–original draft. T. Muramatsu: Supervision. J. Inoue: Supervision. J. Inazawa: Conceptualization, supervision, funding acquisition, writing–review and editing.

This work was supported by KAKENHI (18H02688, 19K07709) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and partially supported by the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from Japan Agency for Medical Research and Development, AMED. We thank Ayako Takahashi and Rumi Mori for technical assistance.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data